We investigated the molecular diversity of the major agricultural pest Bemisia tabaci and of its associated secondary endosymbionts in Greece. Analyzing mitochondrial DNA, we found that the Q1 (=Q west) is predominant. We used eight microsatellite polymorphic markers to study the genetic structure of 37 populations from mainland and insular Greece, collected on different host species from outdoor and protected crops as well as from non-cultivated plants. In some cases, gene flow was found to be low even between populations separated by just a few kilometres. Bayesian analysis identified two main genetic groups, the first encompassing populations from south Crete and the second composed of populations from north Crete, two other Aegean islands and mainland Greece. Genetic differentiation was not correlated with different host plant species or habitat, or greenhouse versus open environment populations. Gene flow significantly decreased with geographic distance, but no isolation by distance existed when only the samples from mainland Greece or only the samples from Crete were considered. The secondary symbionts Wolbachia and Hamiltonella were present at high frequencies while Arsenophonus, Cardinium and Rickettsia were absent from Greek populations. Multilocus sequence typing of Wolbachia identified two Wolbachia strains. These two strains were found together in most of the populations studied but never in the same host individual. Their role on the observed population structure is discussed.