Crown gall is an economically important disease which occur s most frequently on greenhouse-grown roses in Isfahan . The main symptom of the disease is gall formation on the stem or roots. Young galls are light colored and smooth, but as they mature they become dark and woody. The development of galls, results in an overall weakness, reduction in foliage, water stress and loss of vigor of the infected plants. Crown gall is caused by different biovars of a bacterium, Agrobacterium tumefaciens belong to the family Rhizobiaceae, which survives in the soil. The bacterium enters the plant through wounds and subsequently results in tumor formation within a few weeks. The pathogen also sustains systemically inside the plant and spreads through vegetative propagation of rose plants. The infected plants remain symptomless until favorable conditions in greenhouse lead to gall development. Therefore, tracing the contamination source and distinguishing the causal agent in detail is necessary for identification of the origin of infection and its discarding. To do so, the purpose of this study was to identify tumor-inducing Agrobacterium biovar on roses in Isfahan and determine the presence of the same tumorigenic pathogen in soil and host sap. During 2009- 2010, over 40 rose plants with characteristic galls were collected from 10 greenhouses located in Isfahan. Enrichment and plating method on selective media and PCR assays were used for isolation of agrobacteria from surface sterilized galls and checking the presence of agrobacteia in fluidal sap and potting soil mixture of the hosts. According to colony characteristics on TY agar containing antibiotic kanamycin (50 mg/ ml), 27 isolates were initially characterized as Agrobacterium sp. On the basis of tumor inducing capability on geranium, tomato and carrot discs, and biochemical properties, the isolates were identified as Agrobacterium tumefaciens biovar 1. In PCR, the primer pair VCF/VCR amplified a target of 730-bp fragment from the virC operon of the 24 pathogenic strains in this study, but failed to amplify the band in three isolates. The banding pattern of PCR-RFLP of the PCR products from VCF/VCR for all of the isolates were similar indicating their close relatedness. Nucleotide sequence analyses of theVCF/VCR fragment from the representative strain (strain T9) indicated that A.tumefaciens isolates inducing gall formation on greenhouse-grown roses in Isfahan share high similarity with A.tumefaciens strain C58 (biovar 1) and the related agrobacteria previously deposited in the GenBank database. PCR assays using 14vir / 44vir, tms2A/tms2B, virD2 (A-C) and virD2 (A-E) also confirmed that all the isolates belong to A. tumefaciens . The positive PCR results for the RBF-RBR