Unconventional myosins are critical motor proteins in the vertebrate inner ear. Mutations in any one of at least six different myosins can lead to human hereditary deafness, but the precise functions of these proteins in the ear are unknown. This study uses a comparative approach to better understand the role of myosins VI and VIIa in vertebrate ears by examining protein distribution for these two myosins in the ears of evolutionarily diverse fishes and the aquatic clawed toad Xenopus laevis. Both myosins are expressed in the inner ears of all species examined in this study. Myo7a localizes to hair cells, particularly the actin-rich hair bundle, in all species studied. Myo6 also localizes to hair cells, but its distribution differs between species and end organs. Myo6 is found in hair bundles of most fish and frog epithelia examined here but not in anterior and posterior utricular hair bundles of American shad. These results show that myo7a distribution is highly conserved in diverse vertebrates and suggest functional conservation as well. The finding of myo6 in fish and Xenopus hair bundles, however, suggests a novel role for this protein in anamniotic hair cells. The lack of myo6 in specific American shad utricular hair bundles indicates a unique quality of these cells among fishes, perhaps relating to ultrasound detection capability that is found in this species.