Mitochondrial DNA cytochrome c oxidase subunit 1 (DNA barcode) and nuclear microsatellite flanking region sequences were used to analyse populations of putative “sugarbeet root aphid”, Pemphigus betae Doane, from sites in Alberta, Canada. Three sympatric genotypes were revealed, identified as P. betae, P. populivenae Fitch, and an undetermined third species. All three genotypes formed morphologically indistinguishable galls on the same set of cottonwood (Populus L., Salicaceae) host species, often on the same tree. Gall morphology is frequently used to identify Pemphigus species. Our results indicate that this practice may be unreliable for these three taxa at least.