A polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) approach based on the variation in intron sequences of the glucose-6-phosphate dehydrogenase (Zw) gene was used to assess genetic variability in 26 populations and infestations of the Mediterranean fruit fly (medfly), Ceratitis capitata. Beginning with the exon-primed intron-crossing PCR (EPIC–PCR) method to amplify introns of this gene, five alleles were identified on the basis of DNA sequence variants. Several of these variants affect recognition sites for the restriction enzymes RsaI and TaqI. Using these enzymes in successive digestions of the EPIC-PCR products, each of these alleles can be identified directly from individuals. From this, surveys were conducted to document genotypes and allele frequencies in these samples. The relationships of existing populations and the invasion process represented by new infestations of the medfly were analysed using a principal coordinate analysis and the amova method to quantify the distribution of genetic diversity at different levels in a hierarchical manner. From these results, a framework of genetic relationships among the populations and infestations is presented. In addition, for at least some of the infestations, populations that are probably acting as sources of origin have been identified.