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Serious outbreaks of grapevine crown gall disease were observed in major Serbian viticultural regions during the last five years. Tumorigenic Agrobacterium vitis was identified as a causal agent by using conventional bacteriological and molecular tests. The 36 studied strains of A. vitis showed homogeneous biochemical and physiological characteristics, but were heterogeneous in their pathogenic properties, especially on tomato and sunflower. Furthermore, genetic differences related to chromosomal and plasmid DNA were observed. The Ti plasmid of 35 strains was classified as the octopine/cucumopine (O/C) type, whereas one was classified as the vitopine (V) type. The O/C strains were further divided into O/C-1 and O/C-2 groups based on PCR analysis. Moreover, the sequence analysis of the 16S-23S rRNA ITS region provided robust and precise delineation of studied strains. Although a high level of genetic diversity in A. vitis strains from Serbia was revealed by using this approach, their genotypic relatedness with the strains from other countries suggested their common origin. Also, association between the chromosomal and plasmid DNA was determined for some phylogenetic groups and clusters.