Abstract: The present disclosure discloses a method for identifying a M1 generation plant mutant resulting from physical and chemical mutagenesis, a method for acquiring the plant mutant, a mutant gene, and use thereof. The method for identifying a M1 generation plant mutant resulting from physical and chemical mutagenesis includes: mutagenizing a plant to obtain an M1 generation plant mutant, extracting a mixed pool of DNA from the obtained M1 generation plant mutant, subjecting the mixed pool of DNA to high-depth targeted sequencing for a target gene region, and aligning a sequencing result with the target gene region to identify whether there are target single nucleotide polymorphisms (SNPs) and/or Indel. The method of the present disclosure has high efficiency and high accuracy, involves simple operations, and is of progressive significance for the identification and acquisition of innovative germplasms.