An annual bluegrass (Poa annua L.) biotype with limited susceptibility to POST flumioxazin applications was identified in Georgia. The objectives of this research were to quantify tolerance levels of this biotype (R-biotype) to protoporphyrinogen oxidase (PPO) inhibitors and characterize physiological responses to flumioxazin. In dose–response experiments on 3-to 5-tiller plants, flumioxazin and sulfentrazone rates required to reduce dry-shoot biomass 50% from the nontreated were >14.5 and 10.4 times greater for the R-biotype, as compared with a susceptible (S)-biotype, respectively. Establishment of the R-biotype from seed was completely controlled by PRE applications of flumioxazin and oxadiazon, similar to the S-biotype. Tank mixtures of chlorpyrifos with flumioxazin did not enhance biomass reductions of the R-biotype, suggesting that tolerance levels may not be related to cytochrome P450–associated metabolism. In laboratory experiments, the R-biotype averaged 27% less electrolyte leakage, as compared with the S-biotype, after flumioxazin treatments. Lipid peroxidation in the R-biotype, as measured by malondialdehyde levels, averaged 25% less than the S-biotype at 72 h after broadcast flumioxazin treatments at 280 and 560 g ha ^-1. The tolerance to POST applications of PPO inhibitors in this P. annua biotype is associated with less lipid peroxidation and electrolyte leakage as compared with the S-biotype. These biochemical differences in biotypes may contribute to erratic levels of POST control from flumioxazin and could contribute to PPO-inhibitor resistance.