Field inspection indicated that crown gall disease occurs in various fruit-tree-growing areas in Jordan, including Almafraq, Alsalt, Alshobak, Altafila, Alyadoda, Amman, Irbid, Jerash, Jordan Valley and Madaba. Twohundred tumorigenic Agrobacterium isolates were obtained from stone fruit rootstocks (bitter almond, GF677, GF305, Myrobalan, Nemaguard and Mahaleb), pome fruit rootstocks (apple seedling, quince, and clonal rootstock MM106), grapevine, olive, pomegranate, carob, rose and Cichorium pumilum. The pathogenicity of these isolates was proved through artificial inoculation on tomato seedlings and Kalanchöe plants, and through tmr gene detection with PCR. Biochemical and physiological testing of the isolates indicated the occurrence of biovar 1 (60.5%), biovar 2 (23.5%), and biovar 3 (1%), in addition to an intermediate biovar (15%). Seventy-seven percent of isolates were found to be sensitive to agrocin 84; of these, most (66.9%) belonged to biovar 1.