The genetic polymorphism and the biotype identity of the tobacco whitefly Bemisia tabaci (Gennadius) have been studied in population samples taken from different localities within Greece from cultivated plants growing in greenhouses or in open environments and from non-cultivated plants. Two different approaches were used: sequencing of the mitochondrial cytochrome oxidase I (mtCOI) gene and genotyping using microsatellite markers. Analyses of the mtCOI sequences revealed a high homogeneity between the Greek samples which clustered together with Q biotype samples that had been collected from other countries. When genetic polymorphism was examined using six microsatellite markers, the Greek samples, which were all characterized as Q biotype were significantly differentiated from each other and clustered into at least two distinct genetic populations. Moreover, based on the fixed differences revealed by the mtCOI comparison of known B. tabaci biotype sequences, two diagnostic tests for discriminating between Q and B and non-Q/non-B biotypes were developed. Implementation of these diagnostic tools allowed an absence of the B biotype and presence of the Q biotype in the Greek samples to be determined.