Penstemon bicolor is a relatively rare, short-lived perennial herb of conservation concern and is found in the Mojave Desert and surrounding areas of southern Nevada, southeastern California, and northwestern Arizona. Two varieties of P. bicolor are named according to the color of their corolla: variety bicolor, the yellow or pink-colored form, and variety roseus, the magenta-colored form. The rarity of P. bicolor var. bicolor, coupled with its limited distribution, raises concerns about the genetic diversity present in the species, which is critical for the survival of species in the face of environmental stressors, such as habitat loss. Conservation management strategies for P. bicolor rely partially on the taxonomic status of variety bicolor and variety roseus; if these varieties are in fact distinct genetic lineages more appropriately defined as different species, then the implementation and urgency of management strategies would require updating accordingly. We analyzed amplified fragment length polymorphisms and inter-simple sequence repeats for 13 populations of P. bicolor from southern Nevada and northwestern Arizona to assess genetic diversity within the species and to identify the genetic distinctiveness, if any, between varieties. Our findings reveal surprisingly high levels of genetic diversity, in contrast to expectations for rare, perennial, outcrossing plants. Penstemon bicolor does, however, face ongoing threats of habitat destruction and potential local extirpation due to urbanization of the greater Las Vegas area, and we recommend that the current protective status of the species be maintained. Additionally, our analyses of differentiation failed to reveal genetic distinctiveness between varieties; we thus do not recommend that varieties of P. bicolor be elevated to species level.