Dytiscidae is the most diverse family of beetles in which both adults and larvae are aquatic, with examples of extreme morphological and ecological adaptations. Despite continuous attention from systematists and ecologists, existing phylogenetic hypotheses remain unsatisfactory because of limited taxon sampling or low node support. Here we provide a phylogenetic tree inferred from four gene fragments (cox1, rrnL, H3 and SSU, ≈ 4000 aligned base pairs), including 222 species in 116 of 174 known genera and 25 of 26 tribes. We aligned ribosomal genes prior to tree building with parsimony and Bayesian methods using three approaches: progressive pair-wise alignment with refinement, progressive alignment modeling the evolution of indels, and deletion of hypervariable sites. Results were generally congruent across alignment and tree inference methods. Basal relationships were not well defined, although we identified 28 well supported lineages corresponding to recognized tribes or groups of genera, among which the most prominent novel results were the polyphyly of Dytiscinae; the grouping of Pachydrini with Bidessini, Peschetius with Methlini and Coptotomus within Copelatinae; the monophyly of all Australian Hydroporini (Necterosoma group), and their relationship with the Graptodytes and Deronectes groups plus Hygrotini. We found support for a clade formed by Hydroporinae plus Laccophilini, and their sister relationship with Cybistrini and Copelatinae. The tree provided a framework for the analysis of species diversification in Dytiscidae. We found a positive correlation between the number of species in a lineage and the age of the crown group as estimated through a molecular clock approach, but the correlation with the stem age was non-significant. Imbalances between sister clades were significant for several nodes, but the residuals of the regression of species numbers with the crown age of the group identified only Bidessini and the Coptotomus + Agaporomorphus clade as lineages with, respectively, above and below expected levels of species diversity. © The Willi Hennig Society 2008.