AbstractExtraocular muscles are classically grouped as four rectus and two oblique muscles. However, their description and potential associations with species behavior are limited. The objective was to characterize extraocular muscles in four Myliobatoidei rays from diverse habitats with divergent behaviors. Heads (10 per species) of Dasyatis hypostigma, Gymnura altavela, Mobula thurstoni and Pteroplatytrygon violacea were decalcified and dissected to characterize and describe extraocular muscles. Principal component analysis (PCA) was used to evaluate relationships between muscle length and species; for P. violacea, D. hypostigma and G. altavela, these were qualitatively and quantitatively consistent with the general pattern of extraocular muscles in vertebrates. In contrast, for M. thurstoni, the two oblique muscles were completely fused and there was a seventh extraocular muscle, named m. lateral rectus β (both were apparently novel findings in this species). There were also significant differences in eye disposition in the chondrocranium. The PCA axis 1 (rectus muscles) and PCA axis 2 (oblique muscles) accounted for 98.47% of data variability. Extraocular muscles had significant differences in length and important anatomical differences among sampled species that facilitated grouping species according to their life history. In conclusion, extraocular muscles are not uniform in all vertebrate species, thereby providing another basis for comparative studies.