Channel catfish is the primary aquaculture species in the United States. The hybrids catfish generated by crossing female channel catfish (Ictalurus punctatus) and male blue catfish (Ictalurus furcatus) is widely used in production because of its superior performances. In the face of global climate variability, it is of interest to develop catfish strains tolerant to a broader climate conditions such as elevated temperature. In order to identify genes that are associated with thermal stress in channel catfish on a genome scale, we conducted a whole transcriptome profiling using RNA-Seq with a family of 306 hybrid catfish. Samples were collected into three groups: the control group, the intolerant group and the tolerant group after exposure to a higher temperature. Two tissues, liver and gill, were collected and total RNA was isolated for high throughput sequencing using the Illumina HiSeq 2000. Approximately 330 million quality reads were generated, totaling 31,255,426,762 bases. Assembly of the trimmed sequences generated 203,574contigs with an average length of 503 bp. Our preliminary expression analysis identified 1,967 significantly up-regulated genes and 1,754 down-regulated genes associated with heat tolerance in the gill, and 2,160 significantly up-regulated genes and 1,244 down-regulated genes in the liver with a cut-off of two-fold change. Real-time PCR is being conducted on the randomly selected genes to validate the expression results obtained through digital analysis of RNA-Seq. Gene set enrichment analysis and gene pathway analysis are being conducted to identify a set of genes enriched in response to the heat stress. This work should set the foundation for studying heat tolerance and eventually select for more tolerant catfish genotypes for aquaculture.