Simple sequence repeat (SSR) markers were used to construct the DNA fingerprinting database of 27 Seedless Watermelon Major varieties in China and the genetic diversity was also analyzed. 24 polymorphic primer pairs could amplify 66 genotypes, and 2.75 genotypes were detected by each primer pair with the range from 2 to 5. The PIC values ranged from 0.19 to 0.66 with the mean of 0.37. 4 varieties had unique bands. The genetic similarity coefficients of the 27 varieties ranged from 0.7045 to 1.0 with the mean of 0.8683. Besides ‘Zhengkangwuzi No1’and‘Xuefenghuapiwuzi’, the other varieties could be separated clearly from each other. Furthermore, all the varieties were classified into 3 groups, at the level of genetic similarity coefficient 0.83 according to cluster analysis by an un-weighted pair-group average method with arithmetic mean.