Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are members of the voltage-gated channel superfamily and play a critical role in cellular pacemaking. Overall sequence conservation is high throughout the family and channel functions are similar but not identical. Phylogenetic analyses are imperative to understand how these genes have evolved and to make informed comparisons of HCN structure and function. These have been previously limited, however, by the small number of available sequences, from a minimal number of species unevenly distributed over evolutionary time. We have now identified and annotated thirty-one novel genes from invertebrates, urochordates, fish, amphibians, birds and mammals. With increased numbers and a broader representation, a more precise sequence comparison was performed and an evolutionary history for these genes was constructed. Our data confirm the existence of at least four vertebrate paralogs and suggest that these arose via three duplication and diversification events from a single ancestral gene. Additional lineage specific duplications appear to have occurred in urochordate and fish genomes. Based on exon boundary conservation and phylogenetic analyses, we hypothesize that mammalian gene structure was established, and duplication events occurred, after the divergence of urochordates and before the divergence of fish from the tetrapod lineage. In addition, we identified highly conserved sequence regions that are likely important for general HCN functions, as well as regions with differences conserved among each of the individual paralogs. The latter may underlie more subtle isoform-specific properties that were otherwise masked by the high identity among mammalian orthologs and/or inaccurate alignments between paralogs.