Drosophila ananassae and its relatives have many advantages as a model of genetic differentiation and speciation. In this report, we examine evolutionary relationships in the ananassae species subgroup using a multi-locus molecular data set, karyotypes, meiotic chromosome configuration, chromosomal inversions, morphological traits, and patterns of reproductive isolation. We describe several new taxa that are the closest known relatives of D. ananassae. Analysis of Y-chromosomal and mitochondrial haplotypes, shared chromosome arrangements, pre-mating isolation and hybrid male sterility suggests that these taxa represent a recent evolutionary radiation and may experience substantial gene flow. We discuss possible evolutionary histories of these species and give a formal description of one of them as D. parapallidosa Tobari sp. n. The comparative framework established by this study, combined with the recent sequencing of the D. ananassae genome, will facilitate future studies of reproductive isolation, phenotypic variation and genome evolution in this lineage.