This study evaluated the performance and cold tolerance of different Nile tilapia stocks, selected at suboptimal temperature and identified alleles of microsatellite markers associated with cold tolerance. After 67 days of rearing at average temperature of 22.4°C, tilapia juveniles from different stocks were evaluated, tagged with a transponder and acclimated to 16°C. Subsequently, the temperature was reduced 1°C per day until 11°C and from that temperature decreases of 0.5°C per day were made until total mortality. A caudal fin sample was collected, and their genetic profile was characterized through the microsatellite markers UNH879, UNH916 and UNH999. Tilapias of stocks S3, S6 and S7 showed higher growth at suboptimal temperatures, 42 to 47% higher than base population. In the cold tolerance assay, tilapias from stocks S5 and S7 showed lower mean death temperature. Through molecular analysis, it is stated that allele 186 bp of UNH879 and 147 bp of UNH916 were more frequently found in animals cold‐tolerant. Artificial selection for final weight during winter in southern Brazil can assist in the selection of tilapias improved to rearing in suboptimal temperatures. Finally, using microsatellite markers, it is possible to direct selective breeding to obtain tilapias more adapted to low temperatures.