Myosin heavy chains (MHC) are cytoskeletal motor proteins essential to the process of muscle contraction. We have determined the complete sequences of the Xenopus cardiac MHC genes, α-MHC and ventricular MHC (vMHC), and have characterized their developmental expression profiles. Whereas α-MHC is expressed from the earliest stages of cardiac differentiation, vMHC transcripts are not detected until the heart has undergone chamber formation. Early expression of vMHC appears to mark the cardiac conduction system, but expression expands to include the ventricle and outflow tract myocardium during subsequent development. Sequence comparisons, transgenic expression analysis, and comparative genomic studies indicate that Xenopus α-MHC is the true orthologue of the mammalian α-MHC gene. On the other hand, we show that the Xenopus vMHC gene is most closely related to chicken ventricular MHC (vMHC1) not the mammalian β-MHC. Comparative genomic analysis has allowed the detection of a mammalian MHC gene (MyH15) that appears to be the orthologue of vMHC, but evidence suggests that this gene is no longer active. Developmental Dynamics 233:1287–1293, 2005. © 2005 Wiley-Liss, Inc.