Soybean [Glycine max (L.) Merr] is an important crop due to high concentrations of protein and oil. Soybean is an annual diploid plant (2n = 40) having family fabaceae and it belongs to legumes. Most of the commercially grown soybean cultivars contain about 40% protein and 20% oil comprising 85% unsaturated fatty acids (oleic, linoleic and linolenic). These fatty acids (polyunsaturated fatty acids) regulate lipid and cholesterol metabolism and prevent narrowing in artery veins. For those reasons, refined soybean oil is widely used all over the world. Modifying seed oil composition has become a major goal in soybean breeding programs. Genotypes with elevated oleic acid content and reduced linolenic acid content are desirable to improved functionality of soybean oil by increasing oil utility at higher temperatures. Genetic mapping and QTL detection are promising tools to optimize selection in genetic breeding programs, as it allows more accurate study of the genetics of quantitative traits. The knowledge of the genetic variation within accessions from germplasm collections is essential to facilitate the introgression of genes of interest into commercial cultivars. hi present research work, thirteen soybean genotypes were screened by using 15 PUFA specific SSR markers. From 15 SSR markers used in this study, markers AAPTlb (significantly associated with linoleate content and having correlation with increase in oleate content), FAD2-2C (associated with oleate and linoleate content), FAD2-2D (associated with linolenate content), Satt581 (closest marker for QTL qLLE-0 governing for the decreased linoleic acid) and Satt303 (oleate specific) were polymorphic and the other markers AAPTla (associated with oleate and linolenate content), Satt200 (oleate specific), Sattl56 (specific to oleate content), Sattl53 (specific to oleate content), Satt394 (specific to oleate content), Satt356 (specific to oleate content), Satt243 (specific to oleate content), Satt418 (specific to oleate), Satt235 (QTL qLLN-G) and Sattl35 (QTL qLL-Dlb) (closest markers for governing decreased linolenic acid content) were monomorphic. Among the 13 genotypes, genotype JS9752 showed more polymorphism therefore this genotype might be considered for further screening using molecular (QTL and population study) and biochemical analysis of PUFA content using LCMS/ GC-MS. In present investigation we tried to detect primary variants of 13 different soybean genotypes associated with PUFA using SSR markers (specific to PUFA) and the result obtained in this research work might be useful if correlated with biochemical and population studies for MAS breeding for development of soybean lines with desirable fatty acid content. Based on SSR analysis data genetic similarity values ranged from 0.579-1.00 with an average 0.789. Genetic distance values determined using Jaccard’s dissimilarity matrix, were ranged between 0.053-0.421 with an average 0.105. The highest genetic distance value 0.421 was shown by genotype JS9752. The cluster analysis was also performed using SSR analysis data. Two major clusters were categorized as Cluster A and B sharing 67% similarity with each other. The cluster A divided into two sub-cluster and consisted 12 genotypes. Genotype MAUS9752 was separately grouped and found to be unique in cluster B. The total proteins of 13 soybean genotypes were isolated using urea extraction method described by Damania et ah, (1983). The isolated proteins were analyzed using SDS-PAGE method described by Laemmli (1970). The SDS profile of 13 genotypes showed monomorphic banding pattern. Among the 13 genotypes, genotype JS61-2 showed unique banding pattern. The results of seed protein banding patterns could be used as a general biochemical fingerprint for soybean.