Abstract: A preliminary comparative analysis of genetic characteristics between F2 and F3 hybrid populations of Oreochromis niloticus♀ Sarotherodon melanotheron♂ was conducted by using microsatellite markers from the second generation genetic linkage map of Nile tilapia. The results show that 22 pairs of primers were amplified and no polyploidy individual was detected inF2 andF3 populations,seven different amplified loci were screened out in hybridsF2 and F3 populations from28 loci. InF2 hybrid population, average allele was 2. 71; average polymorphic information content was 0. 466; observed heterozygosity was 0. 632. InF3 hybrids, the average allele was 2. 14; average polymorphic information content was 0. 370; observed heterozygosity was 0. 432. The genetic heterozygosity ofF3 population was lower than that ofF2 population. InF3 population, the allele number at four loci was identical withF2 population,while the allele numbers at three loci were less than that ofF2 population. Moreover, there were two homozygous loci inF3 population; the change of alleles inF3 population showed a homozygous trend. The results provide some fundamental data for inheritance of different generations and tilapia hybridization utilization.