Lolium rigidum is the most important weed in Australian agriculture and the pre-emergence dinitroaniline herbicides (e.g. trifluralin) are widely and persistently used for Lolium control. Consequently, resistance evolution to dinitroaniline herbicides has been increasingly reported. Resistance-endowing target-site 𝛼-tubulin gene mutations are identified with varying frequencies. The present study investigated the putative fitness cost associated with the common resistance mutation Val-202-Phe, and the rare resistance mutation of Arg-243-Met causing helical plant growth.