Soybean (Glycine max) is an important legume that is used to fulfill the need of protein and oil of large number of population across the world. There are large numbers of soybean germplasm present in the USDA germplasm resources. Finding and understanding genetically diverse germplasm is a top priority for crop improvement programs. The current study used 20 functional EST-SSR and 80 SSR markers to characterize 96 soybean accessions from diverse geographic backgrounds. Ninety-six of the 100 markers were polymorphic, with 262 alleles (average 2.79 per locus). The molecular markers had an average polymorphic information content (PIC) value of 0.44, with 28 markers ≥ 0.50. The average major allele frequency was 0.57. The observed heterozygosity of the population ranged from 0–0.184 (average 0.02), while the expected heterozygosity ranged from 0.20–0.73 (average 0.51). The lower value for observed heterozygosity than expected heterozygosity suggests the likelihood of a population structure among the germplasm. The phylogenetic analysis and principal coordinate analysis (PCoA) divided the total population into two major groups (G1 and G2), with G1 comprising most of the USA lines and the Australian and Brazilian lines. Furthermore, the phylogenetic analysis and PCoA divided the USA lines into three major clusters without any specific differentiation, supported by the model-based STRUCTURE analysis. Analysis of molecular variance (AMOVA) showed 94% variation among individuals in the total population, with 2% among the populations. For the USA lines, 93% of the variation occurred among individuals, with only 2% among lines from different US states. Pairwise population distance indicated more similarity between the lines from continental America and Australia (189.371) than Asia (199.518). Overall, the 96 soybean lines had a high degree of genetic diversity.