The recent finding of Xylella fastidiosa (Xf) in olive trees in southern Italy, the scanty molecular information on this bacterium and its association with the olive quick decline syndrome (OQDS) prompted the necessity to isolate and acquire more genetic data on the type of strain present in that region. For the first time, the bacterium was isolated from infected olive on culture media. Genetic information were obtained through genomic comparison with other subspecies or strains. The sequences of thirteen genes from its genome, comprising seven housekeeping genes (leuA, petC, lacF, cysG, holC, nuoL and gltT) usually used in multilocus sequence typing (MLST) systems, and six genes involved in different biochemical functions (RNA Pol sigma-70 factor, hypothetical protein HL, 16S rRNA, rfbD, nuoN, and pilU), were analyzed. The sequences of the biochemical function genes were explored individually to study the genetic structure of this bacterium, while the MLST genes were linked together into one concatameric sequence (4161 bp long) to increase the resolution of the phylogenetic analysis when compared with Xf strains previously reported. Sequence analyses of single genes showed that the Xf olive strain is distinct from the four previously defined taxons (Xf subsp. fastidiosa, Xf subsp. multiplex, Xf subsp. sandyi and Xf subsp. pauca) with a dissimilarity rate that reached 4%. In particular, Xf from olive shared the greatest identity with the strain "9a5c" (subsp. pauca), but was nevertheless distinct from it. Similarly, the MLST based on concatameric sequences confirmed the genetic variance of Xf from olive by generating a novel sequence type profile (ST53). Phylogenetic tree analyses showed that Xf from olive clustered in one clade close to subspecies pauca (strains "9a5c" and "CVC0018"), but was nevertheless distinct from them. These results indicate molecular divergence of this olive bacterium with all other strains yet reported.