Variation in mitochondrial DNA (mtDNA) was assessed for >100 populations of Mediterranean fruit flies Ceratitis capitata (Weidemann), collected from the New and Old World using 3 restriction enzymes, EcoRV, MnlI, and XbaI. Previously reported variation for some populations was based on Southern blot analysis of nucleic acid extractions digested with EcoRV and XbaI. MnlI variation for these populations and complete restriction enzyme profiles for many new populations were obtained with a polymerase chain reaction amplification-based method that revealed polymorphism in portions of the mitochondrial genes for NADH dehydrogenase subunits 4 and 5. Highest levels of mtDNA diversity were found in samples from sub-Saharan Africa. Lower levels of mtDNA polymorphism were found in the countries of the Mediterranean region, where the Mediterranean fruit fly has been established for >150 yr. Examination of South and Central American populations revealed a disjunct pattern of mtDNA haplotypes with relatively low intrapopulational variation, reflecting multiple introductions into the New World. Our approach provides improved resolution of possible source countries for historical infestations in the United States.