Temperature tolerance is crucial in aquaculture, as it greatly affects growth, survival, and overall performance. Traditional methods of improving temperature tolerance are often limited by low heritability estimates and the challenge of precisely assessing the trait. This study used low-coverage whole-genome sequencing (lcWGS) with genotype imputation to identify genetic markers linked to cold stress in Nile tilapia. Fish exposed to cold conditions at two sites in Egypt produced a dataset of 7,300,252 filtered SNPs. Out of these, 10 SNPs located in an intronic region of a noncoding RNA (lncRNA) on LG16 were strongly linked to cold stress. Further investigation suggested that these SNPs alter micro-RNA (miRNA) binding sites, potentially affecting interactions with miRNAs miR-133b and miR-139, both of which are associated with stress response and cold adaptation. Validating these markers across various fish populations could support efforts to improve cold tolerance in tilapia through selective breeding.