Parasites are among the most diverse groups of life on Earth, yet complex natural histories often preclude studies of their speciation processes. The biology of parasitic plants facilitates in situ collection of data on both genetic structure and the mechanisms responsible for that structure. Here, we studied the role of mating, dispersal and establishment in host race formation of a parasitic plant. We investigated the population genetics of a vector-borne desert mistletoe (Phoradendron californicum) across two legume host tree species (Senegalia greggii and Prosopis velutina) in the Sonoran desert using microsatellites. Consistent with host race formation, we found strong host-associated genetic structure in sympatry, little genetic variation due to geographic site and weak isolation by distance. We hypothesize that genetic differentiation results from differences in the timing of mistletoe flowering by host species, as we found initial flowering date of individual mistletoes correlated with genetic ancestry. Hybrids with intermediate ancestry were detected genetically. Individuals likely resulting from recent, successful establishment events following dispersal between the host species were detected at frequencies similar to hybrids between host races. Therefore, barriers to gene flow between the host races may have been stronger at mating than at dispersal. We also found higher inbreeding and within-host individual relatedness values for mistletoes on the more rare and isolated host species (S. greggii). Our study spanned spatial scales to address how interactions with both vectors and hosts influence parasitic plant structure with implications for parasite virulence evolution and speciation.