The breeding of herbicide-resistant wheat varieties has helped control weeds in wheat fields economically and effectively. Imidazolinone (IMI) herbicides are popular as they have low toxicity in mammals, are effective at small doses, and exhibit broad-spectrum herbicidal action in the field.herefore, the isolation and genetic and molecular characterization of IMI-resistant wheat mutants will enhance weed management in wheat fields. In the present study, 352 IMI-resistant plants were isolated by genetic screening from a mutant pool prepared by EMS-based random mutagenesis. Cloning of the mutated genes from the IMI-resistant plants indicated that ten taals alleles had been isolated, and mutation in one of three TaALS homolog genes conferred IMI resistance, and such a mutation is a dominant trait. Further analysis showed that taals-d exhibited the greatest IMI resistance, whereas taals-b exhibited the weakest resistance to IMI among three homologous taals mutants. In terms of IMI resistance, the taals triple mutant was stronger than the taals double mutants, and the taals double mutants were stronger than the single mutants, indicating a dose-dependent effect of the TaALS mutation on IMI resistance in wheat. Biochemical analysis indicated that the mutation in TaALS increased the tolerance of TaALS to inhibition by IMI. Our work details the genetic and molecular characterization of als wheat mutants,provides a foundation for understanding IMI resistance and breeding wheat varieties with herbicide resistance, and indicates that genetic screening using a mutagenized pool is an effective and important means of breeding crops with additional desired agricultural traits.