Heat shock proteins (HSPs) consist of a large group of chaperones whose expression is induced by high temperature, hypoxia, infection and a number of other stresses. Among all the HSPs, Hsp40 is the largest HSP family, which bind to Hsp70 ATPase domain in assisting protein folding. In this study, we identified 57 hsp40s in channel catfish (Ictalurus punctatus) through in silico analysis using RNA-Seq and genome databases. These genes can be classified into three different types, Type I, II and III, based on their structural similarities. Phylogenetic and syntenic analyses provided strong evidence in supporting the orthologies of these HSPs. Meta-analyses of RNA-Seq datasets were conducted to analyze expression profile of Hsp40s following bacterial infection. Twenty seven hsp40s were found to be significantly up- or down-regulated in the liver after infection with E. ictaluri; 19 hsp40s were found to be significantly regulated in the intestine after infection with E. ictaluri; and 19 hsp40s were found to be significantly regulated in the gill following infection with F. columnare. Altogether, a total of 42 Hsp40 genes were regulated under disease situations involving three tissues and two bacterial infections. The significant regulated expression of Hsp40 genes after bacterial infection suggested their involvement in disease defenses in catfish.