Switchgrass (Panicum virgatum L.) has gained wider attention due to its recognition and use as a model herbaceous crop species for bioenergy production. Genetic diversity information in lowland switchgrass cultivars can help to specify cultivars to be used in the breeding programs aiming for hybrid vigor. The objective of this research was to analyze genetic variation within and among five lowland switchgrass cultivars using amplified fragment length polymorphism (AFLP) markers. AFLP polymorphisms indicated the presence of high genetic variation within lowland switchgrass cultivars with ‘Alamo’ exhibiting the highest genetic variation and ‘Performer’ the lowest. The Nei’s genetic diversity parameters revealed the lowest genetic distance between cultivars ‘Alamo’ and ‘Cimarron’ and the highest value between cultivars ‘Alamo’ and ‘Kanlow’. ‘Alamo’ and ‘Cimarron’ were clustered together while ‘BoMaster’, ‘Kanlow’, and ‘Performer’ were grouped into the other cluster. In addition, there were clusters with mixed genotypes. The findings of this study can be used to select diverse lines as parents for heterosis and inbreeding studies.