The activation of the complement system is tightly regulated by a group of plasma and cell membrane-associated proteins for host cell protection. In humans, these regulatory protein genes are clustered in a region named the regulator of complement activation (RCA) gene locus and can be categorized into two groups. The group 1 gene cluster has been reported in zebrafish, but information regarding the RCA locus remains scarce in fish. Here we identified two closely linked RCA group 2 genes in zebrafish, ZRC1 and ZRC2, which had all the features characteristic of known RCA group 2 genes. Both ZRC1 and ZRC2 were closely linked to the PFKFB1 gene and located 17 Mkb downstream of the PFKFB2 gene; in contrast, RCA group 2 genes are closely linked to PFKFB2 in frogs, chickens, and humans. However, both the direction of the RCA group 2 genes relative to PFKFB2 and the order of the RCA group 2 gene-encoded proteins in zebrafish were comparable to those in frogs, chickens, and humans. ZRC1 and ZRC2 shared 71.1% identity to each other, implicating that they might have originated by gene duplication after the split of the fish/mammalian common ancestor. Moreover, ZRC1 and ZRC2 encoded a membrane-associated protein and a soluble protein, respectively, and displayed different expression patterns, suggesting that functional divergence has already occurred. This is the first report showing the presence of the RCA group 2 cluster as well as the membrane-associated complement regulatory protein in zebrafish, providing a better understanding of the origin and evolution of RCA proteins.