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 in F₂ and F₃ populations, seven different amplified loci were screened out in hybrids F₂ and F₃ populations from 28 loci. In F₂ hybrid population, average allele was 2.71;average polymorphic information content was 0.466;observed heterozygosity was 0.632. In F₃ hybrids, the average allele was 2.14;average polymorphic information content was 0.370;observed heterozygosity was 0.432. The genetic heterozygosity of F₃ population was lower than that of F₂ population. In F₃ population, the allele number at four loci was identical with F₂ population, while the allele numbers at three loci were less than that of F₂ population. Moreover, there were two homozygous loci in F₃ population; the change of alleles in F₃ population showed a homozygous trend. The results provide some fundamental data for inheritance of different generations and tilapia hybridization utilization.