Wild Mediterranean fruit fly specimens collected from various regions worldwide were screened for the glycine to alanine (Gly->Ala) point mutation (G328A) in the acetylcholinesterase enzyme, presumably causing resistance to organophosphates. We found that the single nucleotide polymorphism (SNP) responsible for this amino acid change is located at the beginning of exon 6 of the Ccace2 gene. The identification of the exact location of the SNP permitted PCR primer design around this site and direct sequencing of the corresponding genomic region. We detected the resistance allele in natural Mediterranean fruit fly populations from Brazil and Spain, but not from other sites in four continents. The known treatment history of sites suggests that the resistance buildup is linked to organophosphate application in the field. The PCR-based detection provides a screening method useful for monitoring Mediterranean fruit fly insecticide resistance in local populations and improving pest management strategies accordingly.