In the zebrafish embryo, two distinct classes of muscle fibers have been described in the forming myotome that arise from topographically separable precursor populations. Based entirely on cross-reactivity with antibodies raised against mammalian and chick myosin heavy chain isoforms slow twitch muscle has been shown to arise exclusively from "adaxial" myoblasts, which migrate from their origin flanking the notochord to form a single layer of subcutaneous differentiated muscle cells. The remainder of the myotome differentiates behind this migration as muscle fibers recognized by anti-fast myosin heavy chain (MyHC) antibodies. To identify unambiguous molecular markers of cell fate in the myotome, we have characterized genes encoding zebrafish fast and slow MyHC. Using phylogenetic and expression analysis, we demonstrate that these genes are definitive molecular markers of slow and fast twitch fates. We also demonstrate that zebrafish embryonic slow twitch muscle co-expresses both slow and fast twitch MyHC isoforms, a property that they share with primary fibers of the amniote myotome.