Habitat fragmentation due to anthropogenic activities is the major cause of biodiversity loss. Endemic and narrowly distributed species are the most susceptible to habitat degradation. Penstemon scariosus is one of many species whose natural habitat is vulnerable to industrialization. All varieties of P. scariosus (P. scariosus var. albifluvis, P. scariosus var. cyanomontanus, P. scariosus var. garrettii, P. scariosus var. scariosus) have small distribution ranges, but only P. scariosus var. albifluvis is being considered for listing under the Endangered Species Act. We used eight microsatellites or simple sequence repeats (SSRs) loci and two amplified fragment length polymorphism (AFLP) primer combinations to investigate the population genetic structure and diversity of P. scariosus varieties. Moreover, we compared the utility of the two marker systems in conservation genetics and estimated an appropriate sample size in population genetic studies. Genetic differentiation among populations based on F st ranged from low to moderate (F st = 0.056-0.157) and from moderate to high when estimated with D es (D es = 0.15-0.32). Also, AMOVA analysis shows that most of the genetic variation is within populations. Inbreeding coefficients (F is) were high in all varieties (0.20-0.56). The Bayesian analysis, STRU CTU RE, identified three clusters from SSR data and four clusters from AFLPs. Clusters were not consistent between marker systems and did not represent the current taxonomy. MEMGENE revealed that a high proportion of the genetic variation is due to geographic distance (R 2 = 0.38, P = 0.001). Comparing the genetic measurements from AFLPs and SSRs, we found that AFLP results were more accurate than SSR results across sample size when populations were larger than 25 individuals. As sample size decreases, the estimates become less stable in both AFLP and SSR datasets. Finally, this study provides insight into the population genetic structure of these varieties, which could be used in conservation efforts.