Drosophila melanogaster originated in Africa, spread to Europe and Asia, and is believed to have colonized the New World in the past few hundred years. Levels of genetic variation are typically reduced in New World populations, consistent with a founder event following range expansion out of Africa and the Old World. We describe the patterns of mtDNA length variation within and among several populations of Drosophila melanogaster from the Old and New World. MtDNA length variation is due to insertion and deletion of tandem repeats in the control region (D-loop) of D. melanogaster mitochondrial genome. The distinct mutational dynamics of this system provide an opportunity to compare the patterns of variation in this marker to those of other markers with different mutational pressures and linkage relationships. The data show significantly more length variation in African and Asian samples than in New World samples. New World samples also show more pronounced skew of the length distribution. Our results are distinct from an earlier study that showed significantly higher levels of length variation and heteroplasmy. The level of heteroplasmy is highly correlated with the number of years that samples have been maintained in laboratory culture, suggesting that relaxed selection in small populations permits the accumulation of mtDNA length variation and heteroplasmy. Together, the data indicate that mtDNA length variants retain a signature of founder events and selection, and suggest that further investigation into the mutation-selection dynamics of the D-loop region of mtDNA would provide a distinct and informative marker for analysis of the recent history of populations.