The objective of this study was to investigate the resistance level of Japanese foxtail to haloxyfop, an acetyl coenzyme A carboxylase (ACCase; EC 6.4.1.2)–inhibiting herbicide. Eleven biotypes were collected from oilseed rape fields in different areas in Jiangsu and Anhui provinces where haloxyfop had been continuously applied for various periods. Biotypes were assessed by two different methods, a seed bioassay and whole-plant assay, to identify the most resistant and susceptible biotypes for further studies on the activity of the target enzyme ACCase. A good correlation was obtained between the two different bioassay methods. The Jurong and Chuzhou biotypes were the most resistant and susceptible biotypes, respectively, whereas the other nine biotypes showed variable and relatively low degrees of haloxyfop resistance. Furthermore, target-site enzyme sensitivity results confirmed that the Jurong biotype was resistant to haloxyfop with a concentration of herbicide causing 50% inhibition of ACCase activity (IC50) of 9.19 µM, whereas the IC50 of the susceptible biotype (Chuzhou) was 0.76 µM, giving a resistance index of 12.