Spotted knapweed, Centaurea stoebe L. ssp. micranthos (Gugler) Hayek (formerly C. maculosa Lam.) (Asteraceae:Cynareae), a non-indigenous invasive perennial forb has been the target of an intensive Integrated Pest Management program utilizing biological control agents. One such biological control organism, Urophora quadrifasciata (Meigen) (Diptera:Tephritidae), the UV knapweed seed-head fly, has become well distributed since its first recorded immigration into the western United States from Canada and from subsequent intentional releases in the eastern United States. While sampling spotted knapweed in eastern Tennessee for the incidence and distribution of U. quadrifasciata, three species of parasitoids associated with U. quadrifasciata were detected. Two species, Pteromalus sp. (Hymenoptera:Pteromalidae) and Brasema sp. (Hymenoptera:Eupelmidae), were located at all six sampling locations. One Eurytoma sp. (Hymenoptera:Eurytomidae) was detected at only one location. This research provides the first documented confirmation of all three parasitoid species associated concurrently with U. quadrifasciata. The negative impact on the development of populations of U. quadrifasciata will be addressed.