In this study, we applied genetic linkage mapping to analyze linkage groups (LG) related to sex determination in five Nile tilapia (Oreochromis niloticus) families and their offspring. Six microsatellite loci (UNH995 and GM258 on LG1; UNH168 and GM354 on LG3; UNH216 and UNH898 on LG23) were used for genotyping. The results showed that two microsatellite loci on LG23 (UNH216 and UNH898) were associated with sex determination (p<0.05) in three Nile tilapia (N1-1, N1-2, and N2-2 family), indicating that the sex determination in Nile tilapia might be influenced by LG23. Furthermore, the empirical results showed that the sex determination system in the N1-1 and N1-2 families was XX-XY. Analysis of the N1 offspring for LG23 indicated that allele D on UNH216 and allele E on UNH898 were associated with male determination. Thus, this study preliminarily confirmed that the genetic markers associated with male determination could be useful for genetic selection and improvement of genetic male Nile tilapia.