Abstract Cytochrome P450 family 4 (CYP4) proteins metabolize fatty acids, eicosanoids, and vitamin D and are important for chemical defense. The purpose of this study was to determine the evolutionary relationships between vertebrate CYP4 subfamilies and raise functional hypotheses regarding CYP4 subfamilies with little empirical data. 132 CYP4 sequences from 28 species were utilized for phylogenetic reconstructions by maximum likelihood and Bayesian inference. Monophyly was not found with the CYP4T and CYP4B subfamilies. CYP4V clustered with invertebrate subfamilies. Evolutionary rates of functional divergence were high in pairwise comparison with CYP4X yet, comparisons with mammalian CYP4F22 genes generally had no statistically significant divergence. Radical biochemical changes were detected in regions associated with substrate binding and the active site in comparisons among the CYP4A, CYP4X, and CYP4B subfamilies. Lastly, gene expression patterns, determined in silico with EST libraries from human, chicken, frog and fish, for CYP4V was markedly different between human and actinopterygian species. Further consideration should be given to the nomenclature of the CYP4T and CYP4B subfamily genes. Strong support was seen for the placement of CYP4A as a basal subfamily to CYP4X and CYP4Z. The B, B′, J′, K′, K″ helices and a region at the end of C-terminus were suggested as conserved regions in CYP4 genes. The function of CYP4X was hypothesized to specialize in metabolism of long chain fatty acids. CYP4F22 genes may share a similar function to other CYP4F genes, although gene expression sites were different.