The Speckled Rattlesnake (Crotalus mitchellii) is a polytypic taxon presently composed of five subspecies that range across southwestern North America, including the Baja Peninsula and islands in the Pacific Ocean and Sea of Cortés. The principles of genealogical concordance were employed to test the taxonomic status of three of the five subspecies (C. m. mitchellii, C. m. pyrrhus, and C. m. stephensi). We used two molecular marker systems: mitochondrial (mt) DNA ATPase 8 and 6 genes (675 base pairs, bp), and introns 5 and 6 of the nuclear (n) DNA ribosomal protein (RP) gene (449 bp). These markers were evaluated across 104 individuals of C. mitchellii: C. m. mitchellii (n  =  3), C. m. pyrrhus (n  =  83), C. m. stephensi (n  =  18), with Sistrurus c. catenatus as the distant outgroup. Deep phylogenetic splits were detected in the subspecies of C. mitchellii, with 5.0–6.4% mtDNA sequence divergence (SD) separating C. m. mitchellii and C. m. pyrrhus, while C. m. mitchellii and C. m. stephensi had SD values of 6.4–7.3%. Similarly, C. m. pyrrhus and C. m. stephensi had SD values of 5.2–6.7%. In addition, C. m. mitchellii and C. m. pyrrhus were identical in all 449 intron bp, but C. m. stephensi differed from both at a single nucleotide polymorphism. Our molecular results diagnose C. m. stephensi as sister to mainland subspecies of the C. mitchellii complex, a result consistent with certain head scalation characters and its northernmost geographic distribution in this complex. Furthermore, four morphological synapomorphies (supraocular scales prominently ridged and/or creased, contact between rostral and prenasal scales, ground coloration of tail congruent with that of body, and black rings in the distal 15% of the tail) also diagnose C. m. stephensi from all other subspecies of C. mitchellii. We hypothesize that the northern distribution of C. m. stephensi likely resulted from two vicariant events: Pliocene expansion of the Sea of Cortés as the Salton Trough, and Pliocene development of the lacustrine Bouse Embayment along the Colorado River drainage. Despite earlier conclusions based on morphology, our molecular results showed no evidence of intergradation between C. m. pyrrhus and C. m. stephensi. Based on the principles of genealogical concordance, we advocate that C. m. stephensi be elevated to a full species, which renders a minimum of two species within the C. mitchellii clades we examined.