Penstemon caryi is a narrow-range endemic angiosperm found primarily on sparsely vegetated limestone outcrops in the Bighorn and Pryor Mountains of north-central Wyoming and south-central Montana. A former candidate for listing under the Endangered Species Act, and currently a species of concern in these states, P. caryi is potentially threatened by habitat loss due to encroaching anthropogenic activities, such as limestone quarrying and road construction. In an effort to assess the capacity of P. caryi to withstand habitat loss and degradation, we used simple sequence repeats (SSRs) and amplified fragment length polymorphism (AFLP) markers to examine genetic diversity and differentiation of P. caryi populations from the Pryor Mountains of Montana and the southern Bighorn Mountains of Wyoming. Our analyses revealed overall moderate to high levels of genetic diversity in P. caryi, but with relatively lower levels of diversity in populations from the Pryor Mountains. There are fewer known individuals of Penstemon caryi in Montana, and because the examined populations from Montana exhibited less genetic diversity than those from Wyoming, populations from Montana may face an increased risk of population decline and extirpation. Both the AFLP and SSR data sets found population structure between different regions (Montana vs. Wyoming), while AFLP markers identified further subdivision between Wyoming populations. Despite moderate to high levels of genetic diversity, P. caryi is still a species at risk due to impending environmental stresses such as global climate change and human encroachment, which may be especially troublesome given its narrow distribution and the specificity of its habitat preferences.