We built TFS sea tilapia strains, but not as good as the market size and growth of the Nile tilapias. Therefore, to improve the seawater adaptability of tilapia, we bred TFS strain and the red TsRn strain from the Nile system, then established forward and diallel crossing F1, inbreeding F2 and first backcross generation BC1F1. Observing the separation of the proportion of seawater (33 psu) adapt to these offsprings, we hypothesize that there are two dominant alleles involved in tilapia in seawater adaptation. Use the establishment of segregation groups and randomized polymorphic DNA (RAPD) technology to select associated gene markers. Using fingerprint analysis of 80 sets of loci, we checked genetic markers with differences and identified 4 RAPD loci with polymorphism, and then further applied Sequence Characterized Amplified Region (SCAR) technology to establish SCAR550, SCAR400, and SCAR300, and proved its polymorphic difference between TFS and TFSxTsRn. This study successfully established a full cross F1 and inbreeding F2 that can be adapted to seawater. In addition, the selection, optimization, and positioning of associated gene markers adapted to seawater can effectively improve the screening efficiency and shorten the breeding time.