In this study, we applied the microsatellite DNA markers and genetic linkage method to analyze the relationship between linkage groups (LG) and sex determination in two blue tilapia (Oreochromis aureus) families. The microsatellite loci (GM201 and UNH104 on LG1; GM271, GM354, UNH131 and UNH168 on LG3) were used for genotyping. The results showed that GM271, GM354, UNH131 and UNH168 were associated with sexuality (p<0.001) in two blue tilapia families (the A1 and A2 families), indicating that the sex-determining locus might be located on LG3 in local blue tilapia. Furthermore, the microsatellite markers on LG3 were closely related with the WZ-ZZ system in the A1 and A2 families. In addition, chromosome crossovers were observed in a small number of offspring. The A2 family was then subjected to quantitative traits loci mapping. In conclusion, the methods we used could be useful for analyzing the sex-determining locus in other tilapia species, and for facilitating the genetic selection and breeding of these fish.