The annual wild soybean (Glycine soja), known as the ancestor of the cultivated soybean (G. max), is endemic to East Asia with most of its range in China. It is believed that the wild soybean holds a reservoir of genetic variation potentially useful for improvement of cultivated soybeans. Our study combined molecular techniques with evaluation of botanical traits to investigate genetic diversity and genetic specificity caused by geographic differentiation in wild soybean populations in China. A total of 196 wild accessions from three distinct geographic regions (Northeast China, Huang-Huai-Hai Valleys and South China, abbreviated as NEC, HHH and SOC, respectively) were genotyped using 52 SSR markers and phenotyped using 10 botanical traits. The average allelic richness (NA) of the entire wild population and Simpson diversity index (H) were 16.1 and 0.852, respectively; higher than those of cultivated populations (NA = 11.4, H = 0.773). Among the three geographic populations, the SOC population had the highest genetic diversity (NA = 12.9, H = 0.842), the NEC population the next highest (NA = 12.4 and H = 0.834), and the HHH population the lowest (NA = 11.4, H = 0.805). Population-specific alleles existed on a number of loci (including AW132402 (Linkage group A2), Satt522 (F), satt150 (M), Sat_332 (D1a), Satt046 (K), sct_190 (K), thus indicating the existence of genetic differentiation among, and ecological specificity of geographic populations. Analysis of botanical traits revealed high variation, diversity (H= 0.710) and geographic differentiation, especially in growth period traits. These differences in botanical traits indicate the significance of natural selection due to geographic variation in day-length and temperature. Analysis of molecular data and botanical traits indicated that the geographic differentiation observed in botanical traits was based on genetic differentiation, and that genetic diversity of the SOC population was higher than the other two populations.