Abstract: Weconducted ananalysis of paternity and relutedness in 3 family groups of Oreochromis niloticus(♀) Sarotherodon melanotheron( ♂ ) hybrids F1with different parental information and differentsizesby microsatellite markers formthe 2nd genetic linkage map of Nile tilapia. The resultsshow therewere 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 the3 family groups was 0.362~0.504, and within each group was 0.245~ 0.316. Thes individual parents in the3 different family groupswere 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 withhigh parent exclusion probability of 99.99%, 99.99% and 99.91%, respectively. Groups A and B contained 3 half-sib families, while group C contained2 pairs of non-sib families or4 half-sib families.