Molecular assays are often implemented by weed scientists for detection of herbicide-resistant individuals; however, the utility of these assays can be limited if multiple mechanisms of evolved resistance exist. Waterhemp resistant to protoporphyrinogen oxidase (PPO)– inhibiting herbicides is conferred by a target-site mutation in PPX2L (a gene coding for PPO), resulting in the loss of a glycine at position 210 (ΔG210). This ΔG210 mutation of PPX2L is the only known mechanism responsible for PPO-inhibitor resistance (PPO-R) in waterhemp from five states (Illinois, Indiana, Iowa, Kansas, and Missouri); however, a limited number of populations have been tested, especially in Illinois. To verify the ubiquity of the ΔG210 in PPO-R waterhemp populations in Illinois, a previously published allele-specific PCR (asPCR) was used for the detection of the ΔG210 mutation to associate this mutation with phenotypic resistance in 94 Illinois waterhemp populations. The ΔG210 mutation was detected in all populations displaying phenotypic resistance to lactofen (220 g ai ha−1), indicating the deletion is likely the only mechanism of resistance. With evidence that the ΔG210 mutation dominates PPO-R waterhemp biotypes, molecular detection techniques have considerable utility. Unfortunately, the previously published asPCR is time consuming, very sensitive to PCR conditions, and requires additional steps to eliminate the possibility of false negatives. To overcome these limitations, a streamlined molecular method using the TaqMan® technique was developed, utilizing allele-specific, fluorescent probes for high-throughput, robust discrimination of each allele (resistant and susceptible) at the 210th amino acid position of PPX2L.