The assembly of myosin into higher order structures is dependent upon accessory factors that are often tissue-specific. UNC-45 acts as such a molecular chaperone for myosin in the nematode Caenorhabditis elegans, in both muscle and non-muscle contexts. Although vertebrates contain homologues of UNC-45, their requirement for muscle function has not been assayed. We identified a zebrafish gene, unc45b, similar to a mammalian unc-45 homologue, expressed exclusively in striated muscle tissue, including the somites, heart and craniofacial muscle. Morpholino-oligonucleotide-mediated knockdown of unc45b results in paralysis and cardiac dysfunction. This paralysis is correlated with a loss of myosin filaments in the sarcomeres of the trunk muscle. Morphants lack circulation, heart looping and display severe cardiac and yolk-sac edema and also demonstrate ventral displacement of several jaw cartilages. Overall, this confirms a role for unc45b in zebrafish motility consistent with a function in myosin thick filament assembly and stability and uncovers novel roles for this gene in the function and morphogenesis of the developing heart and jaw. These results suggest that Unc45b acts as a chaperone that aids in the folding of myosin isoforms required for skeletal, cranial and cardiac muscle contraction.