The Ca²⁺ channel subunits, encoded by CACNB genes 1-4, are Membrane-Associated GUanylate Kinase (MAGUK) proteins. As auxiliary subunits of voltage-gated Ca²⁺ channels, the subunits facilitate membrane trafficking of the pore-forming 1 subunits and regulate voltage-dependent channel gating. In this report, we investigate whether two zebrafish 4 genes, 4.1 and 4.2, have diverged in structure and function over time. Comparative expression analyses indicated that 4.1 and 4.2 were expressed in separable domains within the developing brain and other tissues. Alternative splicing in both genes was subject to differential temporal and spatial regulation, with some organs expressing different subsets of 4.1 and 4.2 transcript variants. We used several genomic tools to identify and compare predicted cDNAs for eight teleost and five tetrapod 4 genes. Teleost species had either one or two 4 paralogs, whereas each tetrapod species contained only one. Teleost 4.1 and 4.2 genes had regions of sequence divergence, but when compared to tetrapod 4s, they exhibited similar exon/intron structure, strong conservation of residues involved in 1 subunit binding, and similar 5' alternative splicing. Phylogenetic results are consistent with the duplicate teleost 4 genes resulting from the teleost whole genome duplication. Following duplication, the 4.1 genes have evolved faster than 4.2 genes. We identified disproportionately large second and third introns in several 4 genes, which we propose may provide regulatory elements contributing to their differential tissue expression. In sum, both mRNA expression data and phylogenetic analysis support the evolutionary divergence of 4.1 and 4.2 subunit function.