We recently reported that masticatory myosin heavy chain (MHC-M) is expressed as the exclusive or predominant MHC isoform in masseter and temporalis muscles of several rodent species, contrary to the prevailing dogma that rodents express almost exclusively MHC isoforms that are typically found in fast limb muscles and not masticatory myosin. We also reported that the same rodent species express the embryonic/atrial isoform of myosin light chain 1 (MLC1E/A) in jaw-closing muscles and not a unique masticatory MLC1 isoform that others have reported as being expressed in jaw-closing muscles of carnivores that express MHC-M. The objective of this study was to test the hypothesis that MLC1E/A is consistently expressed in jaw-closing muscles whenever MHC-M is expressed as the predominant or exclusive MHC isoform. Jaw-closing muscles, fast and slow limb muscles, and cardiac atria and ventricles of 19 species (six Carnivora species, one Primates species, one Chiroptera species, five marsupial species, an alligator and five turtle species) were analyzed using protein gel electrophoresis, immunoblotting, mass spectrometry and RNA sequencing. Gel electrophoresis and immunoblotting indicate that MHC-M is the exclusive or predominant MHC isoform in the jaw-closing muscles of each of the studied species. The results from all of the approaches collectively show that MLC1E/A is exclusively or predominantly expressed in jaw-closing muscles of the same species. We conclude that MLC1E/A is the exclusive or predominant MLC1 isoform that is expressed in jaw-closing muscles of vertebrates that express MHC-M, and that a unique masticatory isoform of MLC1 probably does not exist.