Crown gall, caused by Agrobacterium tumefaciens, first reported on olive in Libya. A partial 16SrRNA gene sequence confirmed the identity of the A. tumefaciens bacterial isolates. The Ti-plasmid (induce tumors formation) is the real reason of their pathogenicity. Meanwhile, the prerequisite for the control of A. tumefaciens is to have a rapid and reliable detection method of pathogenic isolates in order to select healthy plants therefore, we detected two plasmidic genes, virD2 gene of the virulence (vir) region (amplified by virD2A/C and virD2A/E primers), and T-DNA (on tms gene). The PCR amplified bands analysis had a correspondence to the pathogenicity assay (carrot tumorgenicity) and the biochemical determents’ of the isolates genus.