The genetic diversity of Bemisia tabaci was investigated in Southern Italy using the mitochondrial cytochrome oxidase I (COI) gene as molecular marker and sampling whiteflies on cultivated plants, weeds and bushes. Phylogenetic analysis of COI sequences and restriction analysis of COI amplicons were used to genotype whitefly populations. A PCR-RFLP method based on digestion with the endonuclease ApoI was set up to identify the B. tabaci genetic variants so far recorded from the Mediterranean region. In general, biotype Q populations were most frequently collected (76.0% of all sampled populations). In greenhouse districts, 87.0% sampled populations were identified as biotype Q, and 13.0% were assigned to biotype B. Outside the greenhouse districts, the biotype B was never collected, whilst biotype Q populations were found on weeds and on plants cultivated in family gardens in different environments, also located in interior plains or in mountain areas distant from intensive cultivations of whitefly host plants. A new genetic variant unrelated to B and Q biotypes, which was named Ru, was collected on Rubus ulmifolius and grapevine. Phylogenetic analysis of COI sequences shown that Ru haplotypes form a wellsupported clade sister to the clade including the Asian/ Australian major genetic groups and the Italy major genetic group. The closest relative of the Ru clade (10.7% pairwise genetic distance) was the Italy group, with this latter so far including only the haplotypes of the T biotype. These results were discussed in the light of the recent B. tabaci species concept.