Abstract: In order to establish a rapid, acute and effective seed purity detection system of watermelon hybrids, the variety purity of 5 watermelon hybrids was identified, and the molecular fingerprint of parental germplasms was constructed by using SSR molecular markers. With seeds of 5 new watermelon hybrid varieties‘Shenxuan 958’,‘Shuangsebingqilin’,‘Huangjing’,‘Shengnvhong 2’and‘Shengnvhong 3’together with their parents which were bred by researchers of Shanghai Academy of Agricultural Sciences as test materials, total DNA was extracted from root tips of germinating seed by CTAB. Based on the principle of complementary with female parent and male parent of SSR electrophoresis band which was a hybrid variety, the variety purity and the distinctiveness of these watermelon hybrids were identified by analysis of SSR molecular markers. Five pairs of polymorphic primers (primer BVWS00106, BVWS00209, BVWS0036, BVWS01734 and BVWS00826) out of 28 pairs of SSR core primers were selected to make rapid variety purity identification, and the seed purity of these 5 watermelon hybrids were 98%. Compared with results of morphological identification in field, the coincidence rate of the variety purity was higher than 98%, which was obtained by SSR analysis. And the fingerprints of 9 parental germplasms of the 5 watermelon hybrids were constructed by using polymorphic sites of the selected SSR markers. The results showed that the technical system could meet the requirements of rapid variety purity detection and protection of plant variety right of watermelon new hybrids in laboratory.