The Argentinean Chaco region has been considered the center of origin of Drosophila buzzatii in South America because it contains most of the chromosomal polymorphism detected in natural populations. Two hypotheses have been put forward to explain the distribution of D. buzzatii in Brazil, one proposing that it has only recently passively colonized Brazil via human dispersal and the other suggesting that D. buzzatii has actively migrated to Brazil some time ago. Data from chromosomal inversions support recent colonization, whereas data from allozymes and mtDNA variation indicate that D. buzzatii has been in Brazil longer, favoring an active dispersal hypothesis. In our present work we analyzed data on 56 South American flies, mostly from Brazil, sequenced for the 5’ end of the mtDNA COI gene. The combined use of many neutrality tests and phylogeographic methods (e.g. nested clade analysis) indicated high gene flow throughout most of the range of D. buzzatii, although significant population structure was still detected. The high nucleotide diversity in the Northeast region of Brazil and the results from the nested clade analysis suggest that D. buzzatii has been in Brazil longer than proposed by the passive dispersal hypothesis. Our data indicate that D. buzzatii has been distributed throughout Brazil and Argentina since the Quaternary, though more data from different localities and markers need to be gathered to determine how the occupation of South America by D. buzzatii has occurred.