Abstract: New Gift tilapia, GenoMar tilapia, and the hybrid tilapia of Oreochromis niloticus ♀ ×O.aureus ♂ were three major cultured tilapia lines in south China. Identifying these lines was important especially at fry stage. Using amplified fragment length polymorphism(AFLP)technique, we analyzed a total of 60 samples with five primer pairs. Out of 245 AFLP bands, 53.02% were polymorphic. Seven bands from primer pair E-ACA/M-CAT and two bands from E-ACA/M-CAG were found to be specific and could be used to distinguish the three cultured tilapia lines. As for genetic diversity of the three tilapia lines, gene diversity index of New Gift tilapia, GenoMar tilapia, and the hybrid were 0.1605, 0.1595, and 0.2214, respectively. Percent of polymorphic bands and Shannon index presented the similar trend in gene diversity index in these three lines, suggesting that New Gift tilapia was genetically stable, gene diversity index of GenoMar tilapia was a little small.