Chalcidoidea (Hymenoptera) are extremely diverse with more than 23,000 species described and over 500,000 species estimated to exist. This is the first comprehensive phylogenetic analysis of the superfamily based on a molecular analysis of 18S and 28S ribosomal gene regions for 19 families, 72 subfamilies, 343 genera and 649 species. The 56 outgroups are comprised of Ceraphronoidea and most proctotrupomorph families, including Mymarommatidae. Data alignment and the impact of ambiguous regions are explored using a secondary structure analysis and automated (MAFFT) alignments of the core and pairing regions and regions of ambiguous alignment. Both likelihood and parsimony approaches are used to analyze the data. Overall there is no impact of alignment method, and few but substantial differences between likelihood and parsimony approaches. Monophyly of Chalcidoidea and a sister group relationship between Mymaridae and the remaining Chalcidoidea is strongly supported in all analyses. Either Mymarommatoidea or Diaprioidea are the sister group of Chalcidoidea depending on the analysis. Likelihood analyses place Rotoitidae as the sister group of the remaining Chalcidoidea after Mymaridae, whereas parsimony nests them within Chalcidoidea. Some traditional family groups are supported as monophyletic (Agaonidae, Eucharitidae, Encyrtidae, Eulophidae, Leucospidae, Mymaridae, Ormyridae, Signiphoridae, Tanaostigmatidae and Trichogrammatidae). Several other families are paraphyletic (Perilampidae) or polyphyletic (Aphelinidae, Chalcididae, Eupelmidae, Eurytomidae, Pteromalidae, Tetracampidae and Torymidae). Evolutionary scenarios discussed for Chalcidoidea include the evolution of phytophagy, egg parasitism, sternorrhynchan parasitism, hypermetamorphic development and heteronomy.