Introduction: We investigated a) the genetic diversity and population structure, b) the secondary symbiotic bacterial community and c) the frequencies of target site mutations conferring resistance to pyrethroids and organophospates in Q Bemisia tabaci collected from 5 Mediterranean countries. Material and Methods: Polymorphism at seven microsatellite loci and a 676-bp fragment of the cytochrome oxidase I (COI) gene were investigated for the genetic analysis of B. tabaci populations. Simple PCR based detection assays were used for the discrimination of B. tabaci subgroups and the detection of mutations associated with target site insecticide resistance (iAChE, kdr). Secondary symbionts were detected using specific primers. Wolbachia strains were characterized by Multilocus Sequence Typing (MLST) analysis. Results: Analyses of the 676-bp COI fragment distinguished seventeen haplotypes which delineated two groups (Q1 and Q2), within the Q B. tabaci. Both Q1 and Q2 were found in Spain and France, whereas only Q1 was observed in Greece, Morocco and Tunisia. The analyses of the microsatellite loci polymorphism revealed a high level of genetic differentiation even between some neighbouring samples belonging to the same sub-group within a country. In B. tabaci from Greece, Bayesian analysis revealed two main genetic groups within Q1, the first with populations from South Crete, and the second with populations from continental Greece and North Crete. Genetic differentiation was not correlated with host plant species or habitat. The secondary symbionts Wolbachia and Hamiltonella were present at high frequency while Arsenophonus, Cardinium and Rickettsia were absent. MLST analysis identified two Wolbachia strains which were found together in most of the Greek populations but never in the same host individual. The frequency of pyrethroid resistance mutations L925I and T929V in the para sodium channel gene and the organophosphate resistant mutation F331W in the ace1 gene was de