The light–dark cycle is the primarily environmental stimulus responsible for entraining the major circadian clock located within the suprachiasmatic nucleus (SCN), which in turn coordinates various physiological processes in fish, including those in non‐SCN regions of the brain. To better understand differences in gene expression due to the light–dark cycle, we compared the transcriptomes of Pelteobagrus vachellii in non‐SCN regions of the brain in the daytime (12:00) and nighttime (24:00). The two cDNA libraries yielded 217,575,058 and 350,784,004 paired‐end clean reads, and 311,143 unique transcript fragments (unigenes) averaging 600.94 base pairs were assembled. It was possible to annotate ~7,426 (2.39%) unigenes based on homology with all database sequences. Gene expression analysis revealed that 2,406 and 1,665 annotated unigenes were up‐ and downregulated, respectively, with ≥twofold differences between daytime and nighttime. Enrichment analysis of differentially expressed genes predicted the pathways in which they operate, including development, endocrine function, adaptation to environmental changes and immunity. In summary, characterization of daytime and nighttime gene expression patterns in P. vachellii showed that the light–dark cycle coordinates gene expression in non‐SCN regions of the brain.