Biochemical and molecular characteristics of Agrobacterium tumefaciens strains isolated in Tunisia from different host plants were analysed. A collection of 109 isolates was obtained from diseased plants in several nurseries and orchards located in different regions. The majority of strains was isolated from stone fruit trees and belong to the biovar 1. The analysis of opines produced in tumors revealed that the local strain plasmids were of nopaline type. The 16S rRNA gene sequencing showed the presence of different genomic species among the Tunisian collection; but analysis was not enough to undoubtedly assign any isolate to a single genomic species. The multi-loci analysis carried out by BOX-PCR showed that the local isolates were assigned to frequent genomic species G4 (related to the reference strain B6) and G1 (related to reference strain TTT111), but also to rare genomic species G6 and G7 related respectively to Zutra F/1 and RV3.