Molecular assays are often implemented by weed scientists for detection ofherbicide-resistant individuals; however, the utility of these assays can belimited if multiple mechanisms of evolved resistance exist. Waterhempresistant to protoporphyrinogen oxidase (PPO)– inhibiting herbicides isconferred by a target-site mutation in PPX2L (a gene codingfor PPO), resulting in the loss of a glycine at position 210 (ΔG210). ThisΔG210 mutation of PPX2L is the only known mechanismresponsible for PPO-inhibitor resistance (PPO-R) in waterhemp from fivestates (Illinois, Indiana, Iowa, Kansas, and Missouri); however, a limitednumber of populations have been tested, especially in Illinois. To verifythe ubiquity of the ΔG210 in PPO-R waterhemp populations in Illinois, apreviously published allele-specific PCR (asPCR) was used for the detectionof the ΔG210 mutation to associate this mutation with phenotypic resistancein 94 Illinois waterhemp populations. The ΔG210 mutation was detected in allpopulations displaying phenotypic resistance to lactofen (220 g ai ha−1), indicating the deletion is likely the only mechanism ofresistance. With evidence that the ΔG210 mutation dominates PPO-R waterhempbiotypes, molecular detection techniques have considerable utility.Unfortunately, the previously published asPCR is time consuming, verysensitive to PCR conditions, and requires additional steps to eliminate thepossibility of false negatives. To overcome these limitations, a streamlinedmolecular method using the TaqMan® technique was developed, utilizingallele-specific, fluorescent probes for high-throughput, robustdiscrimination of each allele (resistant and susceptible) at the 210th aminoacid position of PPX2L.