The Mediterranean fruit fly, Ceratitis capitata (Wiedemann), considered as one of the most important agricultural pests, is broadly distributed around the world. From its presumed origin in Africa, the fly spread to several areas in Europe and, over the past 100 yr, to Australia and the Americas. We performed an extensive sampling of Mediterranean fruit fly populations in Argentina, covering most fruit-producing areas. The collection was studied using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and sequencing of ND4 and ND5 genes in the mitochondrial genome. We followed the standard system of nomenclature devised previously for designating mitochondrial haplotypes. Our results revealed 1) a high frequency of the already reported AAC and BBB haplotypes; 2) the presence of the AAB haplotype at very low frequency not previously detected in Argentina; 3) a novel point mutation defining two variants of the AAB haplotype (AABA and AABB), and 4) the presence of the AAA haplotype, only in insects of the strain used in the sterile insect technique. A PCR-RFLP assay was developed to differentiate the AABA and AABB haplotypes. The sequence analysis suggests that the AAC haplotype is most likely derived from the AABA haplotype by one nucleotide change. Inferences about the origin of Mediterranean fruit fly populations in this country are discussed.