Fusobacterium nucleatum forms part of the resident microbiota in both oral and extraoral sites in humans and animals. It is also involved in infections in such sites. Despite the genetic heterogeneity within the species, it has been divided into five subspecies, the validities of which have been questioned. In the present study, 44 F. nucleatum isolates were examined at 21 enzyme loci by using the allozyme electrophoretic technique to establish an accurate genetic framework for taxonomic purposes. Three distinct genetic clusters were identified; one cluster consisted exclusively of extraoral isolates, another cluster consisted predominantly of human oral isolates, and the third cluster consisted of a single human oral isolate. Our results highlight the urgent need for extensive biochemical, immunological, and epidemiological studies to accurately define the systematics of the genus fusobacterium based on the framework derived in this study by using 21 independent genetic characteristics.