Understanding patterns of speciation and subsequent gene flow can clarify the evolutionary origins and history of species endemic to a specific geographic region and reveal genetic patterns important for conservation and management of rare species. We chose Dicerandra from the North American Coastal Plain biodiversity hotspot as a model to explore these concepts because of its endemism and the threatened status of most of its species. Using microsatellite-based population-level analyses of 32 populations from four of the annual species (D. linearifolia var. linearifolia, D. linearifolia var. robustior, D. fumella, D. odoratissima, and D. radfordiana), we addressed questions of genetic diversity, population structure, and hybridization. Strong support was found for the species-level recognition of the recently described D. fumella from the Florida panhandle. Dicerandra linearifolia var. linearifolia showed some regional cohesion of populations, but there was no consistent geographic pattern to the clustering of populations. Dicerandra radfordiana showed consistent clustering with proximate populations of D. odoratissima. Given that D. radfordiana is found at the southeastern extreme of the range of D. odoratissima, these populations may represent the early stages of speciation by isolation. While there are morphological and bioclimatic niche distinctions between D. odoratissima and D. radfordiana, there is no molecular support for a distinct D. radfordiana. Overall, there is modest genetic diversity found at the population level for all Dicerandra annuals. Microsatellite data support previously proposed hypotheses of hybridization between D. linearifolia var. linearifolia and D. odoratissima, but do not support such hypotheses for D. fumella and D. linearifolia var. robustior.