Abstract: In order to study genetic relationship and DNA fingerprinting of commercial soybean cultivars and their patental lines in Huaibei Region, Jiangsu, total genomic DNA of 3 high-yielding (Huaidou) soybean cultivars and their 8 core parental lines was extracted by using CTAB method. The genetic similarity coefficients and DNA fingerprinting of the above-mentioned materials were conducted by using SSR markers. 46 pairs of primers were selected and distributed evenly on the whole genome, of which 2 to 5 alleles were detected by each pair of primers. A total of 125 polymorphism fragments were amplified, whose average number was 2.7 per pair of primers. The cluster analysis was conducted by using unweighted pair-group method with arithmetic means (UPGMA). The genetic variation in most of the experimental materials was lower, and their genetic similarity coefficients changed from 0.5781 to 0.9609. The genetic distance between SSR markers were from 0.0391 to 0.4219 and their average value was 0.2773. The fingerprinting of 5 core primers selected from the above primers could be markedly distinguished between the 3 high-yielding (Huaidou) cultivars. These results not only can be used to detect the cultivars (lines) authenticity and protect intellectual property rights, but also to provide theoretical basis and technical support for effective selection of parents to broaden the genetic basis.