To design appropriate strategies for ex situ and in situ conservation of wild soybean and safeguard the biosafety of the wild soybean gene pool when genetically modified soybeans are grown, it is important to understand its genetic characteristics, and to quantify gene flow and kinship within and between neighboring populations. We analyzed 9 pairs of neighboring populations of wild soybeans using 20 pairs of nuclear SSR markers. Results showed that Chinese wild soybean natural populations had outcrossing rates of 0–3.5% and that most populations contained many kinship families. The kinship families could be attributed to the accumulation of outcrossed offspring within populations during the history of population colonization. Wild soybean is very sensitive to environmental selection, which results in genetic differentiation of populations, and the emergence of specific alleles. We used an index &amp;#964; to explain why genetic differences would exist between the pairwise populations; the interpopulation genetic differentiation chiefly consisted in the differences of allele frequencies over the genome. We found long-distance dispersal (1.5 km) of wild soybean seeds in a land ecosystem. There was close correlation between genetic and geographical distance among natural populations of Chinese wild soybean. Within a distance of 50 km, there was greater gene flow when the distance between populations was shorter. These findings have implications for ex situ and in situ conservation in an ecogeographical region, and also for protection of the gene pool from contamination by GM soybeans in wild soybean species.