Alopecurus myosuroides Huds. is a troublesome weed in wheat (Triticum aestivum L.) fields. In 2017, two A. myosuroides populations that survived pyroxsulam treatment at the recommended field rate were collected from winter wheat fields in China. This study was conducted to determine the resistance level and the basis of resistance to pyroxsulam in A. myosuroides. Sequencing of acetolactate synthase (ALS) revealed a Pro-197-Thr mutation in the HB1 population and two mutations (Pro-197-Thr or Trp-574-Leu) in the SD2 population. Purified subpopulations for the Pro-197-Thr and Trp-574-Leu mutations were produced. Whole-plant dose-response tests and in vitro ALS activity assays showed that the purified sHB1 and sSD2 populations displayed moderate resistance (<10-fold) to pyroxsulam compared to the susceptible SD1 population. These results confirmed that A. myosuroides has evolved resistance to pyroxsulam and that the resistance is conferred by the Pro-197-Thr or Trp-574-Leu mutation in ALS.