The genus Mustelus is the most species-rich of the widespread family Triakidae whereby its taxonomy and systematics have been historically challenging. They represent a significant fraction of the shark catches of small-scale fisheries in the Gulf of California. In order to provide information useful for their management and conservation, the morphological and genetic distinction of the four species found in the northern Gulf of California (M. albipinnis, M. californicus, M. henlei and M. lunulatus) were analyzed. Discriminant analysis of 10 morphometric variables placed each species in a distinct and significantly different region of multivariate morpho-space. The variables contributing most to their distinction were inter-nostril width, mouth length, upper and lower labial furrow length, and mouth width. Restriction fragment length polymorphisms (PCR-RFLP) of the nuclear ITS2 ribosomal DNA (rDNA) confirmed that each species represents a genetically cohesive and independent evolutionary lineage. In spite of the difficulty in differentiating these Mustelus species, a few cephalic measurements are useful to separate them. A PCR-RFLP assay (using RsaI and MspI on ITS2 rDNA amplicons) is also proposed for the molecular differentiation of these commercially harvested smooth-hound sharks, constituting the first molecular marker available for their identification. These data provide morphological and genetic tools that can be used to improve their management and conservation.