Trillium (Melanthiaceae) is a highly diverse genus in the Southeast and includes many local endemics. Previous studies on Trillium in eastern North America identified significant genetic structure associated with limited seed dispersal and historical landscape barriers. In this study, genetic structure was examined in Trillium cuneatum and T. stamineum across Mississippi and western Alabama to look for further evidence that landscape features influence genetic structure at local scales. DNA sequence variation in the trnL intron and trnL-trnF intergenic region was examined across 12 populations of T. cuneatum and six populations of T. stamineum. Samples of Trillium ludovicianum and Trillium foetidissimum were included for comparison. Five and four haplotypes were discovered in T. cuneatum and T. stamineum, respectively, but most populations were fixed for a single haplotype. Haplotypes from T. cuneatum in southwestern Mississippi were also present in congeneric species. Significant geographic structure was found in both species, and divergent haplotypes found on either side of the Tombigbee and Black Warrior Rivers suggest barriers to gene flow in the study area. Strong population differentiation suggests that seed dispersal is limited in both species. Given the strong degree of genetic structure detected in both species and their preference of mesic forests, it is expected that both species will continue to diverge at local scales.