Resistance to protoporphyrinogen oxidase (PPO)-inhibiting herbicides in waterhemp has been shown previously to be the result of a unique mechanism. Specifically, a three–base-pair (3-bp) deletion in the PPX2L gene, a gene encoding both plastid- and mitochondria-targeted PPO enzymes, confers herbicide resistance in this species. Furthermore, when this unique mechanism was initially characterized it was presumed that waterhemp contained three PPX genes, PPX1, PPX2S, and PPX2L, and that the resistant biotypes were missing PPX2S. Here, allele testing and examination of genetic sequence data demonstrate that there are likely only two PPX genes in waterhemp, PPX1 and PPX2L. Next, to determine the prevalence of this mechanism of resistance in Illinois waterhemp, we developed an allele-specific polymerase chain reaction (PCR) marker that amplifies only the 3-bp deletion allele, ΔG210, of PPX2L. By utilizing this marker, we show that the ΔG210 PPX2L allele correlated with whole-plant resistance to PPO inhibitors in each of four other waterhemp populations evaluated from Illinois.