The body plan of Drosophila, and presumably that of other insects, develops under the control of anterior–posterior and dorsal–ventral axes, but no evidence for a left–right axis has yet been found. We used geometric morphometrics to study the wings in three species of flies: Drosophila melanogaster,Musca domestica and Glossina palpalis gambiensis. In all three species, we found that both size and shape showed subtle, but statistically significant directional asymmetry. For size, these asymmetries were somewhat inconsistent within and between species, but for shape, highly significant directional asymmetry was found in all samples examined. These systematic left–right differences imply the existence of a left–right axis that conveys distinct positional identities to the wing imaginal discs on either body side. Hence, the wing discs of Drosophila may be a new model to study the developmental genetics of left–right asymmetry. The asymmetries of shape were similar among species, suggesting that directional asymmetry has been evolutionarily conserved since the three lineages diverged. We discuss the implications of this evolutionary conservatism in conjunction with results from earlier studies that showed a lack of genetic variation for directional asymmetry in Drosophila.