Abstract: We conducted an analysis of paternity and relatedness in 3 family groups of Oreochromis niloticus (♀)× Sarotherodon melanotheron (♂) hybrids F₁ with different parental information and different sizes by microsatellite markers from the 2^nd genetic linkage map of Nile tilapia. The results show there were 20 loci with different-sized alleles in O.niloticus and S.melanotheron detected in 86 pairs of microsatellites, including 13 specific loci and 7 shared allelic loci. The average number of alleles in F₁ was 2.90 and of polymorphism information was 0.439. The genetic distance among the 3 family groups was 0.362~0.504, and within each group was 0.245~0.316. The individual parents in the 3 different family groups were effectively identified by 3 completely higher polymorphic markers combinations. Using genotypic information, 4, 8 and 12 loci were needed for accurate paternity identification in family group A, B and C with high parent exclusion probability of 99.99%, 99.99% and 99.91%, respectively. Groups A and B contained 3 half-sib families, while group C contained 2 pairs of non-sib families or 4 half-sib families.