Drosophila melanogaster and its close relatives are used extensively in comparative biology. Despite the importance of phylogenetic information for such studies, relationships between some melanogaster species group members are unclear due to conflicting phylogenetic signals at different loci. In this study, we use twelve nuclear loci (eleven coding and one non-coding) to assess the degree of phylogenetic incongruence in this model system. We focus on two nodes: (1) The node joining the D. erecta-D. orena, D. melanogaster-D. simulans, and D. yakuba-D. teissieri lineages, and (2) The node joining the lineages leading to the melanogaster, takahashii, and eugracilis subgroups. We find limited evidence for incongruence at the first node; our data, as well as those of several previous studies, strongly support monophyly of a clade consisting of D. erecta-D. orena and D. yakuba-D. teissieri. By contrast, using likelihood based tests of congruence, we find robust evidence for topological incongruence at the second node. Different loci support different relationships among the melanogaster, takahashii and eugracilis subgroups, and the observed incongruence is not easily attributable to homoplasy, non-equilibrium base composition, or positive selection on a subset of loci. We argue that lineage sorting in the common ancestor of these three subgroups is the most plausible explanation for our observations. Such lineage sorting may lead to biased estimation of tree topology and evolutionary rates, and may confound inferences of positive selection.Drosophila melanogaster and its relatives have been used extensively in studies of genetic and morphological variation within and between species. For example, inferences concerning the relative roles of drift, purifying selection, and positive selection in shaping patterns of genetic variation in D. melanogaster often benefit from comparisons to the closely related species D. simulans and D. yakuba (e.g., McDonald and Kreitman, 1991). Similarly, comparative morphologists have used D. melanogaster and its relatives to study the evolution of a number of traits, e.g., genital morphology (Kopp and True, 2002a) and pigmentation (Wittkopp et al., 2002;Prud'homme et al., 2006).Opportunities for, and interest in, using the genus Drosophila in comparative biology is likely to grow in the near future. The availability of complete genome sequences for twelve members of the genus Drosophila (http://species.flybase.net), as well as for several other dipterans, promises to facilitate genome scale studies of molecular evolution. These comparative data will allow for the detection of functionally important genomic regions, as indicated by high levels of conservation or by the signature of positive, diversifying selection. Moreover, the application of genetic and transgenic techniques developed in D. melanogaster to other species will facilitate studies of evolution and development. *Corresponding author: Address: Department of Molecular Biology and Genetics, Biotechnology Building, Co...