Chamaecrista fasciculata is a widely distributed, phenotypically variable species in the eastern U.S. Whereas studies have demonstrated genetic structure and local adaptation in northern areas of its distribution, there has been no comparison of genetic variability among populations at the southern extent where phenotypic variation is more complex. We characterized genetic variation at 14 microsatellite loci for populations in Mississippi and Alabama and compared this to variation in a phenotypic trait, leaf pubescence. Geographic distance, climatic variables, and elevation were evaluated as factors to explain the observed patterns of genetic diversity. A significant amount of variation (19%) resided among populations, but most variation (68%) was among individuals. Assignment of individuals into genetic groups suggests two primary clusters, but these groups are not concordant with known geographical or ecological breaks, nor phenotypic variants. Genetic structure at a regional scale can be characterized as isolation by distance, while environmental factors may play a secondary role in limiting gene flow at local scales. Mean population FST is strongly associated with allelic diversity and heterozygosity, suggesting that genetic drift influences population variation. Despite the presence of genetic and phenotypic variation in southern populations of C. fasciculata, the lack of concordant patterns between these types of variation indicate that they are not driven by the same factors. This study demonstrates how local factors differentially influence the maintenance of intraspecific variation and suggest the southern distributional range is an active area of evolution for C. fasciculata.