The genetic diversity analysis of 137 wild soybean accessions from three populations of Shandong province were analyzed by allele-special DNA markers. One dominant allele was detected at E1 and E4 loci, respectively, while the haplotypes E2-in and E3-Ha were found to be predominant at E2 and E3 loci, respectively. Out of five genotypic combinations, the genotype carrying E1E2-dlE3-HaE4 was predominant. By further analyzing the diversity among the three populations, four genotypic combinations were detected in Linyi population. The genotypes carrying E1E2-inE3-MiE4 were often detected, while E2-in and E3-Mi were major alleles at E2 and E3 loci, respectively. Three genotypic combinations were observed in both Penglai and Rongcheng populations, while E1E2-dlE3-HaE4 was often detected. The alleles E2-dl and E3-Ha were superiority allele on E2 and E3 loci, respectively. A similarity on genetic diversity was found between Rongcheng and Penglai populations, while both were differing with Linyi population that represented a highest genetic diversity. Taken together, the results gained from this study might provide insights for the utilization of wild soybean germplasm resources in Shandong Province.