The study was carried out at Molecular Biology Lab., Plant Genetic Resources Centre of Bangladesh Agricultural Research Institute, Gazipur, Bangladesh. One hundred and fifteen collected germplasm were planted for morphological characterization in the year 2014-15. Young leaves sample has been collected from 90-95 days mature plants based on their morphological traits. The collection of leaf samples was made from morphologically homozygous plants. A total of 96 germplasm of chilli representing different geographical distributions were selected for the present study (Annexure 01). Bulked DNA was isolated from 4-5 fresh leaves using two standard protocols for chilli genomic DNA isolation of which one was SDS (Sodium dodecyl sulfate) and phenol: chloroform: IAA followed by alcohol precipitation described by Saghai-Maroof et al. (1984) and also used by Rahman et al. (2007) with some modifications and another one CTAB (Cetyl trimethylammonium bromide). The quality and quantity of DNA were authenticated by gel electrophoresis and spectrophotometry. If a single high-molecular-weight band was formed, the DNA quality was considered good, and if it formed a smear, the DNA quality was considered to be poor. The modified SDS and phenol: chloroform: IAA protocol found to be cost-effective and latex-free high-quality genomic DNA was obtained successfully compared to the CTAB DNA isolation procedure. Hence, excluding usage of CTAB and liquid nitrogen the modified protocol included digestion with homogenization buffer (Solution: Tris-50 mM, EDTA-25 mM, NaCl-300 mM, 1% SDS and deionized water) at 65ºC for 30 min, extraction with phenol: chloroform: isoamyl alcohol (25:24:1), precipitation with ice-cold and extra pure isopropyl alcohol and purification with absolute ethanol (Plus sodium acetate, 3M) and 70% ethanol chronologically. Finally, DNA sample of each chilli germplasm dissolving in 30-40 μl of TE buffer within 1.5 ml eppendorf tube was preserved separately at -20ºC. The presence of genomic DNA was confirmed on 1% agarose gel qualitatively. The gels were visualized under UV light and photographed using a photo documentation system (UV Transilluminator, Uvitec, UK). All of the DNA samples were found to be in good quality in this study. The electropherogram of DNA of 24 individuals (12 of SDS and 12 of CTAB protocol) from 12 chilli germplasm are shown in Figure 1. The amount of genomic DNA was quantified at 260nm spectrophotometrically (Spectronic GENESYS 10 Bio). Using the absorbance reading obtained for DNA sample of each chilli germplasm, the original DNA concentrations were determined. Identification and selection of microsatellite/SSR primers: Fifty microsatellite primer pairs were identified and those are located at 12 chromosomes from a different publication. Preliminarily, twenty-eight primer pairs of the different chromosomes were tested for their better responsiveness in amplifying the target genomic region of template DNA and to check the expected PCR product sizes in base pairs (Table 1). All 28 primers pairs were shown better responsiveness with clear and expected amplified product sizes and were used for microsatellite analysis in the present study. The Polymerase chain reactions were set up 10 μl volumes containing 50 ng template DNA, 5X Green GoTaq® Reaction Buffer included 7.5 mM MgSO4, 1.25 U/μl Taq DNA polymerase, 0.4 mM each of the dNTPs, 10 μM of each of primer, 0.5% DMSO and a suitable amount of sterile deionized water. The reaction was performed in an oil-free eppendorf Mastercycler nexus Gradient thermal cycler. SSRs were amplified under the following “touchdown” PCR conditions: 94oC/3 min denaturation, 11 cycles of 94oC/0.5 min, 58-60oC/1 min, decreasing by 1oC per cycle, and 72oC/1 min; 30 cycles of 94oC/0.5 min, 52-55-oC/1 min and 72oC/1 min; a final extension for 5 min. For checking amplification, the PCR products were electrophoretically resolved on 2% agarose gel in 1X TBE. If the primer was shown good band resolution intensity, less searing, amplifying the target genomic region of template DNA, the PCR protocol was considered to be correct. The electropherogram of PCR products of 20 primer pairs in two chilli germplasm is shown in Figure 3.The Polymerase chain reactions were set up 10 μl volumes containing 50 ng template DNA, 5X Green GoTaq® Reaction Buffer included 7.5 mM MgSO4, 1.25 U/μl Taq DNA polymerase, 0.4 mM each of the dNTPs, 10 μM of each of primer, 0.5% DMSO and a suitable amount of sterile deionized water. The reaction was performed in an oil-free eppendorf Mastercycler nexus Gradient thermal cycler. SSRs were amplified under the following “touchdown” PCR conditions: 94oC/3 min denaturation, 11 cycles of 94oC/0.5 min, 58-60oC/1 min, decreasing by 1oC per cycle, and 72oC/1 min; 30 cycles of 94oC/0.5 min, 52-55-oC/1 min and 72oC/1 min; a final extension for 5 min. For checking amplification, the PCR products were electrophoretically resolved on 2% agarose gel in 1X TBE. If the primer was shown good band resolution intensity, less searing, amplifying the target genomic region of template DNA, the PCR protocol was considered to be correct. The electropherogram of PCR products of 20 primer pairs in two chilli germplasm. PCR-products were electrophoresed on a 5% denaturing polyacrylamide gel containing 19:1 acrylamide: bis-acrylamide, 10X TBE buffer, 10% APS and ultrapureTemed. Electrophoresis was done using the Triple Wide Mini-Vertical Electrophoresis System, MGV-202-33 (CBS Scientific, USA). Run the gel at 80-90V and 20ºC temperature maintained by a cooling system (Julabo, Germany) upon loading of PCR products for a specified period of time depending on the size of amplified DNA fragment (usually 1 hour for 100 bp). After completion of electrophoresis, the gel was stained with Ethidium bromide. The individual bands in the glass plate were stained and scored for analysis. Scoring and Analysis of Microsatellite Data: The bands representing particular alleles at the microsatellite loci were scored manually and designated the bands as A, B, C, etc. from the top to the bottom of the gel. The genotypes of different individuals were hypothetically scored as AA, BB, CC, etc. for homozygous or as AB, AC, BC etc. for heterozygous. A single genotypic data matrix was constructed for all loci. Polymorphism Information Content (PIC) was computed by deducting the sum of square values for all the frequencies of different alleles produced by a single marker locus from one using the formula: PIC= 1- ΣXi2, Where, Xi is the frequency of the i-th allele of a particular locus. PIC provides an estimate of the discriminatory power of a marker by taking into account, not only the number of alleles that are expressed but also the relative frequencies of those alleles. PIC values range from 0 (monomorphic) to 1 (very high discriminative, with many alleles in equal frequencies). The software DNA FRAG version 3.03 was used to estimate allelic length (Nash, 1991). Expected (He) and observed heterozygosity (Ho) were also calculated as per Nei (1972) formula and with the help of POPGENE (version 1.31) (Yeh et al., 1999) computer package program. Estimation of Nei’s genetic distance values (D) (Nei, 1972) and construction of UPGMA (Unweighted Pair Group Method of Arithmetic Means) dendrogram was constructed using the software POPGENE (Version 1.31) (Yeh et al., 1999).