Mexican red-kneed tarantulas of the genus Brachypelma are regarded as some of the most desirable invertebrate pets, and although bred in captivity, they continue to be smuggled out of the wild in large numbers. Species are often difficult to identify based solely on morphology, therefore prompt and accurate identification is required for adequate protection. Thus, we explored the applicability of using COI-based DNA barcoding as a complementary identification tool. Brachypelma smithi (F. O. Pickard-Cambridge, 1897) and Brachypelma hamorii Tesmongt, Cleton & Verdez, 1997 are redescribed, and their morphological differences defined. Brachypelma annitha is proposed as a new synonym of B. smithi. The current distribution of red-kneed tarantulas shows that the Balsas River basin may act as a geographical barrier. Morphological and molecular evidence are concordant and together provide robust hypotheses for delimiting Mexican red-kneed tarantula species. DNA barcoding of these tarantulas is further shown to be useful for species-level identification and for potentially preventing black market trade in these spiders. As a Convention on International Trade in Endangered Species (CITES) listing does not protect habitat, or control wildlife management or human interactions with organisms, it is important to support environmental conservation activities to provide an alternative income for local communities and to avoid damage to wildlife populations.