Phytomyza Falle´n is the largest genus of leaf-mining flies (Agromyzidae), with over 530 described species. Species of the superficially similar genus Chromatomyia Hardy have been included in Phytomyza by some authors and the status of the genus remains uncertain. Using 3076 bp of DNA sequence from three genes [cytochrome oxidase I (COI), CAD (rudimentary), phosphogluconate dehydrogenase (PGD)] and 113 exemplar species, we identified and tested the monophyly of host-associated species groups in Phytomyza and Chromatomyia and investigated the phylogenetic relationships among these groups. Chromatomyia is polyphyletic and nested largely within Phytomyza; two small groups of species, however, are related more closely to Ptochomyza and Napomyza.