Weeds are a major factor affecting rice yield and quality, and breeding herbicide-resistant rice varieties is an economical and effective approach for weed control. Mesotrione is an HPPD-inhibiting herbicide with great application potential because of its broad weed-control spectrum, safety to succeeding crops, and the low likelihood of resistance development in weeds. Natural alleles conferring mesotrione resistance are present in rice germplasm resources. To identify resistant germplasm, this study employed a liquid culture-based evaluation method to conduct large-scale screening of 760 rice germplasm accessions. Based on resistance phenotypes, the accessions were classified into five grades: 1, 3, 5, 7, and 9. As a result, four grade-1 resistant accessions, 28 grade-3 resistant accessions, and 19 grade-9 highly susceptible accessions were identified. Further field spraying experiments at the seedling and tillering stages showed that the highly resistant germplasm screened in this study could tolerate up to 80 mL/667 m2 of 15% mesotrione suspension concentrate spray in field test. The liquid culture mesotrione resistance identification method established in this study has the advantages of controllable conditions, good uniformity, high efficiency, and scalability, enabling rapid and large scale evaluation of mesotrione resistance in rice germplasm resources. The highly resistant and highly susceptible germplasm identified in this study not only provide excellent genetic resources for breeding new mesotrione-resistant rice varieties, but also offer important materials for resistance gene mining and genetic mechanism analysis.