Simple SummarySoybean is a prominent grain and oil crop in the world. The production of soybean is threatened by weed competition. Recently, CRISPR/Cas9-mediated base editing, which can cause irreversible nucleotide changes in target loci, has been widely used in basic plant science and breeding. However, until now, herbicide-resistant traits in soybean have not been created by the base editor. The objective of this study was to modify different AHAS homologous alleles by base editor to generate a novel AHAS-inhibiting herbicide-resistant soybean. Our results showed that the GmAHAS4 P180S mutants have apparent resistance to AHAS-inhibiting herbicides, which will be beneficial for weed control in soybean production.AbstractWeeds cause the largest yield loss in soybean production. The development of herbicide-resistant soybean germplasm is of great significance for weed control and yield improvement. In this study, we used the cytosine base editor (BE3) to develop novel herbicide-resistant soybean. We have successfully introduced base substitutions inGmAHAS3andGmAHAS4and obtained a heritable transgene-free soybean with homozygous P180S mutation inGmAHAS4. The GmAHAS4 P180S mutants have apparent resistance to chlorsulfuron, flucarbazone-sodium, and flumetsulam. In particular, the resistance to chlorsulfuron was more than 100 times that of with wild type TL-1. The agronomic performance of the GmAHAS4 P180S mutants showed no significant differences to TL-1 under natural growth conditions. In addition, we developed allele-specific PCR markers for the GmAHAS4 P180S mutants, which can easily discriminate homozygous, heterozygous mutants, and wild-type plants. This study demonstrates a feasible and effective way to generate herbicide-resistant soybean by using CRISPR/Cas9-mediated base editing.