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The Chiricahua Mountains in southeastern Arizona are renowned for their exceptional biodiversity and high levels of endemism. Morphological, genomic, behavioral, and distributional data were used to report the discovery of a remarkable new tarantula species from this range.Aphonopelma jacobiisp. nov.inhabits high-elevation mixed conifer forests in these mountains, but also co-occurs and shares its breeding period withA. chiricahua—a related member of theMarxispecies group—in mid-elevation Madrean evergreen oak and pine-oak woodlands. This marks the first documented case of syntopy between two montane endemics in the Madrean Archipelago and adds to our knowledge of this threatened region’s unmatched tarantula diversity in the United States. An emended diagnosis and redescription forA. chiricahuaare also provided based on several newly acquired and accurately identified specimens. Phylogenetic analyses of mitochondrial and genomic-scale data reveal thatA. jacobiisp. nov.is more closely related toA. marxi, a species primarily distributed on the Colorado Plateau, than toA. chiricahuaor the other Madrean Sky Island taxa. These data provide the evolutionary framework for better understanding the region’s complex biogeographic history (e.g., biotic assembly of the Chiricahua Mountains) and conservation of these spiders.