Base-editing-library-induced high density nucleotide substitutions have been applied to screen functional mutations in plants. However, due to limitations in the scope and conversion specificity of base editors, many desired mutations at pivotal protein sites may be overlooked. Here, we developed a prime-editing-library-mediated saturation mutagenesis (PLSM) method to substantially increase the diversity of amino acid substitutions at target sites for in planta screening. At six conserved residues of OsACC1, 16 types of herbicide-resistance-endowing mutations were identified. Most of these mutations exhibit reliable tolerance to aryloxyphenoxypropionate herbicides and have not been reported or applied in rice breeding. In addition, the advantage of PLSM was further shown by comparing the base-editing-mediated mutagenesis at the selected targets. The PLSM method established in this study has great potential for the direct evolution of genes related to agronomically important traits for crop improvement.