Nucleotide sequences of portions of the mitochondrial 12S and 16S ribosomal RNA genes were used to extend a recent study of anuran phylogeny (Hayet al., Mol. Biol. Evol.12: 928–937, 1995) and to further evaluate phylogenetic relationships within the Neobatrachia. An analysis of almost 900 nucleotides from each of 8 new representatives of the Dendrobatidae, Hylidae, Leptodactylidae, and Myobatrachidae, plus 14 available members of the Neobatrachia provides support for 2 major lineages (Bufonoidea and Ranoidea) within this anuran suborder. The neotropical Bufonoidea and their derivatives are monophyletic. There is an interesting association of the 2 Australian myobatrachids with the South African Heleophrynidae, and the Sooglossidae is one of the basal bufonoid lineages. Within the New World bufonoid frogs, a monophyletic Dendrobatidae is strongly supported. An Australian hylid (Pelodryadinae) shows close affinity with the South American hylid Phyllomedusinae. A group composed of Hylinae (HylaandSmilisca), Centrolenidae, Bufonidae, and the hylid Hemiphractinae, with the latter two clustered, was supported significantly. The addition of new taxa has more clearly defined some relationships within the suborder Neobatrachia and has indicated that the families Hylidae, Leptodactylidae, and Myobatrachidae may not be monophyletic.