The northern Mexican Pacific (NMP), the Gulf of California (GC), and Baja California have been recognized as an ecological and evolutionarily dynamic region having experienced significant tectonic and climatic changes leading to the diversification of terrestrial and marine biotas. Zapteryx exasperata is a predominant ray caught in the artisanal fisheries of the NMP. Morphometric and reproductive differences between rays from the GC and the Pacific coast of Baja California (PCBC) regions suggest the presence of distinct populations. We investigate whether this distinction correlates with differences in genetic diversity and differentiation using sequences of the mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 2 (ND2) gene and the noncoding control region (CR) in 63 specimens. Contrary to our expectations, ND2 bore significantly more diversity (h = 0.76) than CR (h = 0.39). Geographic patterns of diversity of CR were opposite to those of ND2, with GC being significantly less (ND2) and more (CR) diverse than PCBC. The diversity of concatenated haplotypes was high (h = 0.84). Low nucleotide diversity suggests the recent coancestry of haplotypes. Marked genetic structure (Φst = 0.23, P < 0.0001) revealed the existence of reproductive isolation and limited matrilineal gene flow between GC and PCBC, which correlates with their phenotypic distinction. These results suggest the influence of factors such as female reproductive philopatry, and ecological or historical vicariant barriers to gene flow. Our results point to the existence of a distinct management unit of banded guitarfish in each region, and add to the increasing evidence attesting to the diversifying nature of this evolutionarily dynamic region.