Italian ryegrass [Lolium perenne L. ssp. multiflorum (Lam.) Husnot], a pernicious weed in wheat fields, has evolved severe resistance to the acetolactate synthase (ALS)-inhibiting herbicides, like pyroxsulam. Here, the derived cleaved amplified polymorphic sequence (dCAPS) markers were developed to detect two distinct mutations at the 197th position of the ALS gene. The method was used to examine and purify the resistant population. Homozygous populations with different 197 site mutations from the same population were obtained, and the target-site resistance mechanisms were investigated. Whole-plant dose-response bioassays show that the resistance index (RI) of the Pro-197-Thr mutant population to pyroxsulam was 508.92, whereas that of the Pro-197-Gln population was 9.75. Similar trends were observed for different herbicides within same mode of action. In vitro ALS assays demonstrated that the Pro-197-Thr population exhibited lower sensitivity to pyroxsulam than the Pro-197-Gln population, consistent with plant bioassays. Furthermore, ALS gene expression of the Pro-197-Thr population analysis is significantly higher than that in the Pro-197-Gln population which may also explain why the Pro-197-Thr population exhibits higher resistance level than the Pro-197-Gln population. Our findings suggest that different amino acid substitutions at one ALS gene locus can confer herbicide resistance with different levels in L. perenne ssp. multiflorum. This study provides valuable insights into the mechanisms of herbicide resistance in L. perenne ssp. multiflorum.