Relationships among members of Ericaceae subfamily Monotropoideae have been difficult to resolve due to reduction and convergent evolution in these parasitic plants. All species in this subfamily are fully mycoheterotrophic, meaning they obtain nutrients by parasitizing fungi rather than through photosynthesis. Here, we examine relationships and host specificity in one of the most widespread species in this subfamily, Monotropa uniflora. We use several lines of evidence to investigate whether there is support for recognizing a segregate, M. brittonii, as distinct. Based on molecular and morphological analysis of Monotropa collected throughout its range in the United States, we find two distinct lineages, one of which corresponds morphologically and geographically to Small's M. brittonii. We identified several morphological characters that differ between the two species. We also observed a high degree of fungal host specificity in M. brittonii, which appears to parasitize almost exclusively Lactifluus subgenus Lactariopsis section Albati. Additionally, M. brittonii was primarily collected from Florida scrub, which are xeric, shrub-dominated habitats that differ substantially from the mesic forests where M. uniflora typically occurs. Based on these molecular, morphological, and ecological differences, we support recognition of M. brittonii as distinct from M. uniflora.