Fluazifop-P-butyl, a selective graminicide, has been widely used to control annual grass weeds for more than three decades in Taiwan, and a resistant (R) biotype of goosegrass has consequently appeared. In this study, liquid chromatography/tandem mass spectrometry (LC/MS/MS) was applied to analyze metabolites of fluazifop-P-butyl in a R biotype of goosegrass. Six signals, including mass-to-charge ratios (m/z) 512, 432, 423, 415, 314, and 160, in positive scanning mode, and four signals, including m/z 788, 623, 593, and 162, in negative scanning mode, were found in the metabolites of the R and S biotypes, respectively. All of the signals of these metabolites in the R biotype showed higher intensity than those of the S biotype, except for m/z 162. Based on the molecular weights of the fragments (MS2 signal) from the molecules (MS1 signal) and comparison with the Metabolite Link Metabolomics database, MassBank database, and related references, one reduced form of fluazifop acid, i.e., 2-[4-(5- trifluoromethyl-2-pyridyloxy) phenoxy] propanol (MW 313); two types of intermediates of MW 163, i.e., 5-trifluoromethyl-2-pyridone and 5-trifluoromethyl-2-hydroxypyridine (or 2-hydroxy-5-trifluoromethyl pyridine); and five possible conjugated compounds containing a common core fragment (MW 255) from fluazifop acid were identified. In addition, another compound, likely degraded from one of the five conjugated compounds, was also detected. Accordingly, the metabolic pathway of fluazifop-P-butyl in goosegrass is described in this study. An enzyme kinetic study on glutathione S-transferase showed that the R biotype has higher affinities to the substrates reduced glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene, with S/R K
ratios of 3.0 and 2.4, respectively. No difference in V
max was found, revealing that the S biotype has a strong ability to bind GSH and herbicide or target molecules and showed susceptibility to fluazifop.