Abstract. Mealybugs (Hemiptera: Pseudococcidae) are small, plant-sucking insects which comprise the second largest family of scale insects (Coccoidea). Relationships among many pseudococcid genera are poorly known and there is no stable higher level classification. Here we review previous hypotheses on relationships and classification and present the first comprehensive phylogenetic study of the Pseudococcidae based on analysis of nucleotide sequence data. We used three nuclear genes, comprising two noncontiguous fragments of elongation factor 1α (EF-1α 5′ and EF-1α 3′), fragments of the D2 and D10 expansion regions of the large subunit ribosomal DNA gene (28S), and a region of the small subunit ribosomal DNA gene (18S). We sampled sixty-four species of mealybug belonging to thirty-five genera and representing each of the five subfamilies which had been recognized previously, and included four species of Puto (Putoidae) and one species each of Aclerda (Aclerdidae) and Icerya (Margarodidae), using Icerya as the most distant outgroup. A combined analysis of all data found three major clades of mealybugs which we equate to the subfamilies Pseudococcinae, Phenacoccinae and Rhizoecinae. Within Pseudococcinae, we recognize the tribes Pseudococcini (for Pseudococcus, Dysmicoccus, Trionymus and a few smaller genera), Planococcini (for Planococcus and possibly Planococcoides) and Trabutinini (represented by a diverse range of genera, including Amonostherium, Antonina, Balanococcus, Nipaecoccus and non-African Paracoccus), as well as the Ferrisia group (for Ferrisia and Anisococcus), some ungrouped African taxa (Grewiacoccus, Paracoccus, Paraputo and Vryburgia), Chaetococcus bambusae and Maconellicoccus. The ‘legless’ mealybugs Antonina and Chaetococcus were not closely related and thus we confirmed that the Sphaerococcinae as presently constituted is polyphyletic. In our analyses, the subfamily Phenacoccinae was represented by just Phenacoccus and Heliococcus. The hypogeic mealybugs of the Rhizoecinae usually formed a monophyletic group sister to all other taxa. Our molecular data also suggest that the genera Pseudococcus, Dysmicoccus, Nipaecoccus and Paracoccus are not monophyletic (probably polyphyletic) and that Phenacoccus may be paraphyletic, but further sampling of species and genes is required. We compare our phylogenetic results with published information on the intracellular endosymbionts of mealybugs and hypothesize that the subfamily Pseudococcinae may be characterized by the possession of β-Proteobacteria (primary endosymbionts) capable of intracellular symbiosis with γ-Proteobacteria (secondary endosymbionts). Furthermore, our data suggest that the identities of the secondary endosymbionts may be useful in inferring mealybug relationships. Finally, cloning polymerase chain reaction products showed that paralogous copies of EF-1α were present in at least three taxa. Unlike the situation in Apis and Drosophila, the paralogues could not be distinguished by either the presence/absence or position of an intron.