Resistance in waterhemp to herbicides that inhibit protoporphyrinogen oxidase (PPO) previously was shown to result from the deletion of a glycine codon at position 210 (ΔG210) in the PPO-encoding gene, PPX2. Research was conducted to determine if this same mechanism accounted for resistance in geographically separated populations—from Illinois, Kansas, and Missouri—and, if so, to determine if the mutation conferring resistance was independently selected. A dose–response study with lactofen indicated that the resistant populations had different levels of resistance. These differences, however, could be accounted for by different frequencies of resistant individuals within populations and, therefore, the dose–response data were consistent with the hypothesis that the populations contained the same resistance mechanism. Direct evidence in support of this hypothesis was provided by DNA sequencing, which showed that nearly all resistant plants evaluated contained the ΔG210 mutation. A variable region of the PPX2 gene was sequenced and resulting sequences were aligned and organized into a phylogenetic tree. The phylogenetic tree did not reveal clear clustering by either geography or phenotype (resistant vs. sensitive). Possibly recombination within the PPX2 gene has masked its evolutionary history.