Pennisetum purpureum Schum. (napiergrass) is a perennial grass used for forage especially in South America and Africa. Over the last 30 years, a USDA–ARS nursery containing accessions collected from all over the world has been established in Tifton, Georgia. The study reported here was conducted to assess the molecular genetic variation and genetic relatedness among 89 accessions from the Tifton nursery using amplified fragment length polymorphism markers, morphological data and ploidy level. Using 218 polymorphic markers from eight selective primer combinations, the 89 accessions were clustered into five groups using a principal components analysis and a dendrogram based on Dice similarity estimates and unweighted pair group method with arithmetic average clustering. These five groups include three groups collected from Kenya, a group from Puerto Rico, and accessions derived from the cultivar Merkeron. This research provides the first molecular characterization of the Tifton nursery, displays the relationships between accessions, and provides potential heterotic groups for napiergrass and pearl millet (Pennisetum glaucum (L.) R. Br.) breeding improvement.