Despite the prevalence of developmental myopathies resulting from muscle fiber defects, the earliest stages of myogenesis remain poorly understood. Unc45b is a molecular chaperone that mediates the folding of thick-filament myosin during sarcomere formation; however, Unc45b may also mediate specific functions of non-muscle myosins (NMMs). unc45b Mutants have specific defects in striated muscle development, which include myocyte detachment indicative of dysfunctional adhesion complex formation. Given the necessity for non-muscle myosin function in the formation of adhesion complexes and premyofibril templates, we tested the hypothesis that the unc45b mutant phenotype is not mediated solely by interaction with muscle myosin heavy chain (mMHC). We used the advantages of a transparent zebrafish embryo to determine the temporal and spatial patterns of expression for unc45b, non-muscle myosins and mMHC in developing somites. We also examined the formation of myocyte attachment complexes (costameres) in wild-type and unc45b mutant embryos. Our results demonstrate co-expression and co-regulation of Unc45b and NMM in myogenic tissue several hours before any muscle myosin heavy chain is expressed. We also note deficiencies in the localization of costamere components and NMM in unc45b mutants that is consistent with an NMM-mediated role for Unc45b during early myogenesis. This represents a novel role for Unc45b in the earliest stages of muscle development that is independent of muscle mMHC folding.