Two exotic crane fly species, Tipula (Tipula) paludosa Meigen (Diptera: Tipulidae) and Tipula (Tipula) oleracea L. have spread considerably in North America beyond original areas of detection in eastern and western Canada. These species are endemic in Europe, and pests in pastures and cereals. The two species differ in life cycles and periods when they feed, warranting species-specific control. Identification presents a challenge because the larvae are not easily distinguishable from each other, and resemble native nonpestiferous species present in sympatry. We collected crane flies from urban landscapes and agricultural fields in Oregon in the western United States. Using polymerase chain reaction (PCR), we sequenced a portion of the mitochondrial cytB gene in 55 individuals (eight adults and 47 larvae) from 29 sites. We observed 7% divergence between exotic and native species. Phylogenetic analysis, using Nephrotoma ferruginea F. as an outgroup, resolved four well-supported monophyletic groups: the exotics, T. (Tipula) oleracea and T. (Tipula) paludosa, and two natives, Tipula (Serratipula) tristis Doane and Tipula (Triplicitipula) sp. Nucleotide divergence between T. oleracea and T. paludosa was P = 0.071, whereas within species divergence was very low (P = 0.0018 and P = 0.0022, respectively). The study indicated that mitochondrial cytB sequences provided an accurate, rapid, and economic technique for separation of T. oleracea and T. paludosa from each other and from native species, and insights on their habitats. The technique will facilitate early pest management decisions, and studies on host plants and geographic distribution, as the two exotic species extend their ranges across North America.