The origins of arthropods and the phylogenetic relationships among their three major living groups (atelocerates, crustaceans and chelicerates) are vigorously contended. To help resolve this, we determined mitochondrial gene arrangements for a chelicerate, a myriapod, two crustaceans, an onychophoran, a mollusc and an annelid, and compared them with published gene orders of other species. The result strongly supports the monophyly of Arthropoda and of Mandibulata (atelocerates plus crustaceans) and refutes the Uniramia (atelocerates plus onychophorans). Gene arrangement comparisons are emerging as a powerful new tool for resolving ancient phylogenetic relationships.