Sarcomeric myosin heavy chain (MyHC) is the major contractile protein of striated muscle. Six tandemly linked skeletal MyHC genes on chromosome 17 and two cardiac MyHC genes on chromosome 14 have been previously described in the human genome. We report the identification of three novel human sarcomeric MyHC genes on chromosomes 3, 7, and 20, which are notable for their atypical size and intron-exon structure. Two of the encoded proteins are structurally most like the slow-beta MyHC, whereas the third one is closest to the adult fast IIb isoform. Data from pairwise comparisons of aligned coding sequences imply the existence of ancestral genomes with four sarcomeric genes before the emergence of a dedicated smooth muscle MyHC gene. To further address the evolutionary relationships of the distinct sarcomeric and nonsarcomeric rod sequences, we have identified and further annotated human genomic DNA sequences corresponding to 14 class-II MyHCs. An extensive analysis provides a timeline for intron gain and loss, gene contraction and expansion, and gene conversion among genes encoding class-II myosins. One of the novel human genes is found to have introns at positions shared only with the molluscan catchin/MyHC gene, providing evidence for the structure of a pre-Cambrian ancestral gene.