Low temperatures limit the development of Oreochromis niloticus (tilapia), and an increase in low-temperature tolerance would increase yields. We studied the responses of 48 individuals of tilapia to low temperatures. The fish were labeled CK, AA, BB, and CC based on treatment (25, 12/1 h, 12/24 h, and 12/48 h, respectively) with CK being the control group. Transcriptome sequencing of 12 individuals yielded 585.51 million clean reads, and at least 0.8326 of the genes were mapped to the reference genome. Comparative analysis revealed 12,448 genes with significantly differential expression, including 792, 1,827, 1,924 upregulated genes and 992, 3,056, 3,857 genes downregulated for AA, BB, and CC, respectively. Differentially expressed genes (DEGs) were validated using RT-PCR for five genes. Functional annotation analysis of the DEGs identified functions associated with response to low-temperature stress. When tilapia were subjected to low-temperature stress, expression changes occurred in genes associated with cytokine-cytokine receptor interaction, metabolic pathways, cell adhesion molecules, material transport, and immunity.