By comparative genomics based on chromosome synteny between human and fish, IL-22, IL-26 and two IFN-gamma-like genes have been isolated in fish. Interleukin-22 and -26 have been identified for the first time outside of mammals and their organization and synteny demonstrates that this cluster of cytokines is well conserved during evolution. The cloned zebrafish IL-22 and -26 homologs are 161 and 169-aa long, respectively. Both the genes harbor a well-conserved IL-10-family signature in their respective C-terminus. The coding regions of these genes are spread in five exons and at the same position as in human homologs. Furthermore, surprisingly two IFN-gamma-like genes are present in tandem, 7.0 kb apart from each other, along this locus. IFN-gamma1 and 2 genes are composed of 170- and 185-aa and share a low amino acid identity of 17.0%. The genomic structures of the isolated fish IFN-gamma genes were similar to its mammalian homologs, sharing a four exon and three phase 0 introns. The presence two IFN-gamma genes in both zebra and pufferfishes demonstrate that the duplication of IFN-gamma might have occurred prior to the teleostean split. The structural homology is further validated by phylogenetic analysis showing that the isolated cytokine genes are homologous to mammalian counterparts. Expression analysis of IL-22, IL-26 and two IFN-gamma genes suggests an active role of these genes in immune responses in fish.