Four species of the genus Rhagoletis are native to Chile: R. nova (Schiner), R. conversa, (Brèthes), R. penela Foote and R. tomatis Foote. Currently, identification of these species is based on morphological criteria, but their strong similarity makes precise recognition difficult. To clarify species separation for quarantine purposes, a reliable method based on a PCR–RFLP procedure is reported. A DNA region containing mitochondrial NADH dehydrogenase genes was selected as a target sequence for the analysis. The amplification products (c. 1 kb) were digested with either SspI or DdeI, yielding specific patterns that differentiated each of the endemic species. Complete nucleotide sequences were determined, confirming empirical restriction maps. This report updates information on the geographical distribution of Rhagoletis species in Chile.