Black spots could be induced in red tilapia at wintering stage. This body color variation causes significant economic loss to the aquaculture industry of red tilapia. Understanding the genetic architecture of wintering black spot variation is of great significance for the selection of pure red tilapia lines. In this study, 37 SNPs and five QTLs controlling for the wintering black spot trait at genome-wide significance were successfully identified on Chr2:4.6–4.9 Mb, Chr2:20.0 Mb, Chr3:30.1–34.0 Mb, Chr8:4.0–6.1 Mb, Chr12:5.9–7.5 Mb in red tilapia (N = 287) by using a genome-wide association study. These QTLs were further verified by genotyping four novel microsatellite markers in the mapping population. Three candidate genes (sema-4c, uvssa and htr2cl1) in these QTL regions showed significantly differentiated expression in response to low temperature (LT) or UVB or UVB + LT challenges in brain or skin tissues of red tilapia as revealed by qRT-PCR analysis. Interestingly, the expression levels of the three candidate genes were more significantly changed under the UVB+ LT stressors than the LT or UVB stress solely. These suggested that the wintering black spots in red tilapia may be regulated by comprehensive environmental factors. •37 SNPs associated with wintering black spot trait were successfully identified in red tilapia.•QTLs were verified in the mapping population using four microsatellite markers.•Three candidate genes showed significant responses to challenges in red tilapia.