Drosophila differens, endemic to Molokai, Drosophila planitibia of Maui, and Drosophila silvestris and Drosophila heteroneura from the island of Hawaii are chromosomally homosequential species that presumably have colonized the newer islands of the Hawaiian archipelago by sequential founder events. We have examined the phylogenetic relationships of these four species by using mitochondrial DNA restriction site data for 23 enzymes. Both distance and character-state analyses indicate that a sequential or monotonic branching relationship exists for mtDNA restriction site data from the four species. The mtDNA data suggest that the maternal lineage that gave rise to D. differens is ancestral to the D. planitibia maternal lineage, which in turn shares the most recent common ancestor with the D. silvestris and D. heteroneura maternal lineages [with Drosophila hemipeza (Oahu) and Drosophila neopicta (Molokai and Maui) as outside references]. We also discuss the phylogenetic implications of the mtDNA data in comparison with other sources of phylogenetic data. We conclude that hybridization of the species in this group has been an important factor in the evolution of the nuclear genomes. Because of small population sizes and mating asymmetries, it is possible that the nuclear genetic distance of species that are physically capable of hybridizing (e.g., on the same island or island complex) is depressed. Consequently the mtDNA genetic distance appears to be more sensitive in establishing the sequence of evolutionary events responsible for the present distribution and population structure of these species.