BACKGROUND: Clomazone is a potent herbicide for controlling weeds that have evolved resistance to other herbicides due to its unique mode of action. Clomazone is used in rice cultivation, but is limited to long-grain cultivars because other cultivars are highly sensitive to it. In this study, we investigated the mechanism of clomazone tolerance in a long-grain cultivar. RESULTS: The long-grain cultivar Kasalath tolerated approximately five-fold higher doses of clomazone compared to two short-grain cultivars, Nipponbare and Koshihikari. While Arabidopsis thaliana transformed with a rice cytochrome P450, CYP81A6, showed resistance to clomazone, the cyp81a6 knockout Kasalath was unchanged in its clomazone sensitivity. The inheritance of clomazone sensitivity in the F1 and F2 of Kasalath and Nipponbare indicated the involvement of multiple loci for clomazone tolerance. Four chromosome segment substitution lines of Nipponbare/Kasalath and Koshihikari/Kasalath exhibited partial tolerance to clomazone. The overlapping DNA region among the four lines is on chromosome 5 within 11.5 Mb. CONCLUSION: Multiple loci are involved in clomazone tolerance in Kasalath, one of which is located on chromosome 5. This information will help develop short-grain cultivars tolerant to clomazone. © 2021 Society of Chemical Industry.