Breeding herbicide tolerance into new cultivars can improve safety and weed control in turfgrass systems. The sensitivity to fluazifop-P-butyl of 27 zoysiagrass (Zoysia spp.) lines was screened under greenhouse conditions to identify potential tolerant germplasm for breeding programs. The herbicide rate that caused 50% biomass reduction (GR₅₀) and the rate that caused 50% injury (ID₅₀) were calculated to select the three most-tolerant and the five most-susceptible lines for studying the physiological mechanisms responsible for fluazifop-P-butyl tolerance. The differences in GR₅₀ and ID₅₀ between susceptible and tolerant lines ranged from 4-fold to more than 10-fold. Cytochrome P450–mediated metabolism was not detected in fluazifop-P-butyl–tolerant lines. Sequencing of the ACCase gene confirmed that none of the seven previously reported mutations conferring resistance to acetyl-CoA carboxylase (ACCase)-inhibiting herbicides in other species were present in any of the tolerant or susceptible zoysiagrass lines studied. An Ala-2073-Thr substitution was identified in two tolerant lines, but thismutation did not completely explain the tolerant phenotype. No clear differences in absorption and translocation rates of ¹⁴C-radiolabeled fluazifop-P-butyl were observed among most lines, with the exception of a susceptible line that exhibited greater translocation than two of the tolerant lines. Metabolite profiles did not differ between tolerant and susceptible lines. Our results suggest that the diversity in tolerance to fluazifop-P-butyl in zoysiagrass germplasmis most likely the result of a combination of different, minor, additive non–target site mechanisms such as translocation rate and compartmentation after absorption.