Striga gesnerioides is a root hemiparasite of wild and cultivated legumes, among which cowpea (Vigna unguiculata) and Indigofera hirsuta are suitable hosts. In this study, we examined the genetic structure and host-parasite interaction of a strain of S. gesnerioides parasitizing I. hirsuta (SGFL) from central Florida (United States). Amplified fragment length polymorphism analysis was used to compare genetic profiles from 71 individual S. gesnerioides plants (SGFL) representing four different populations in central Florida. Our results showed that these individuals are genetically similar, with pairwise genetic distances ranging from 0.00 to 0.037. A cluster analysis grouped all four S. gesnerioides populations from Florida, separating them from S. gesnerioides isolates parasitic on I. hirsuta and cowpea collected from West Africa. One EcoRI and MseI selective primer combination generated a 510-bp fragment present in individuals from the SGFL and the West African isolate parasitic on I. hirsuta, but absent in isolates parasitic on cowpea. Germination of seed from individuals of all four populations of S. gesnerioides parasitic on I. hirsuta from Florida was stimulated by root exudates from cowpea genotypes Blackeye and TVX-3236, known to be highly susceptible to all races of S. gesnerioides parasitic on cowpea in West Africa. SGFL seedlings failed to parasitize cowpea, with the development of attached SGFL seedlings arrested at the tubercle stage. The very high level of genetic uniformity observed within and among the central Florida populations suggests that there was likely a single introduction of the parasite or strong host-driven selection to genetic uniformity.These findings are important in assessing the potential of the parasite as an agronomically significant pest in the United States.