Interleukin-1beta (IL-1beta) is one of the pivotal early response pro-inflammatory cytokines that enables organisms to respond to infection and induces a cascade of reactions leading to inflammation. In spite of its importance and two decades of studies in the mammalian species, genes encoding IL-1beta were not identified from non-mammalian species until recently. Recent research, particularly with genomic approaches, has led to sequencing of IL-1beta from many species. Clinical studies also suggested IL-1beta as an immunoregulatory molecule potentially useful for enhancing vaccination. However, no IL-1beta genes have been identified from channel catfish, the primary aquaculture species from the United States. In this study, we identified two distinct cDNAs encoding catfish IL-1beta. Their encoding genes were identified, sequenced, and characterized. The catfish IL-1beta genes were assigned to bacterial artificial chromosome (BAC) clones. Genomic studies indicated that the IL-1beta genes were tandemly duplicated on the same chromosome. Phylogenetic analysis of various IL-1beta genes indicated the possibility of recent species-specific gene duplications in channel catfish, and perhaps also in swine and carp. Expression analysis indicated that both IL-1beta genes were expressed, but exhibited distinct expression profiles in various catfish tissues, and after bacterial infection with Edwardsiella ictaluri.