Polytene chromosomes banding patterns of ten of the 16 species of the Neotropical annulimana group of Drosophila were used to propose phylogenetic relationships among species. Drosophila annulimana chromosomes were used as the standard sequence and the most parsimonious series of changes (paracentric inversions) were considered. In some cases, intermediate hypothetical rearrangements were proposed to explain the sequences present in a given species. A total of 47 paracentric inversions were detected, most of them (44.7%) in chromosome 4. Three subgroups, partially coincident with those previously proposed based on morphological and karyotypical analyses, were classified as: 1) annulimana subgroup (Drosophila annulimana, D. aracataca, D. aragua, and D. arauna), 2) gibberosa subgroup (D. ararama, D. gibberosa, D. pseudotalamancana, and D. schineri), and 3) arassari subgroup (D. arapuan, and D. arassari).