Objective: Chamaecrista fasciculata is a widespread annual legume across Eastern North America, with potential as a restoration planting, biofuel crop, and genetic model for non-papillinoid legumes. As a non-Papilinoid, C. fasciculata, belongs to the Caesalpiniod group in which nodulation likely arose independently of the nodulation in Papilinoid and Mimosoid legumes. Thus, C. fasciculata is an attractive model system for legume evolution. In this study, we describe population structure and genetic diversity among 32 USDA germplasm accessions of C. fasciculata using 317 AFLP markers developed from 12 primer pairs, to assess where geographically there is the most genetic variation. Results: We found that the C. fasciculata germplasm collection fall into four clusters with admixture among them. After correcting for outliers, our analysis shows two primary groups across Eastern and Central North America. To better understand the population biology of this species, further sampling of the full range of this widespread species is needed across North America, as well as the development of a larger set of markers providing denser coverage of the genome. Further sampling will help clarify geographical relationships in this widespread temperate species.