BACKGROUND: Lolium rigidum is the most important weed in Australian agriculture and pre-emergence dinitroaniline herbicides (e.g., trifluralin) are widely and persistently used for Lolium control. Consequently, evolution of resistance to dinitroaniline herbicides has been increasingly reported. Resistance-endowing target-site α-tubulin gene mutations are identified with varying frequency. This study investigated the putative fitness cost associated with the common resistance mutation Val–202–Phe and the rare resistance mutation Arg–243–Met causing helical plant growth. RESULTS: Results showed a deleterious effect of Arg–243–Met on fitness when plants are homozygous for this mutation. This was evidenced as high plant mortality, severely diminished root and aboveground vegetative growth (lower relative growth rate), and very poor fecundity compared with the wild-type, which led to a nearly lethal fitness cost of >99.9% in competition with a wheat crop. A fitness penalty in vegetative growth was evident, but to a much lesser extent, in plants heterozygous for the Arg–243–Met mutation. By contrast, plants possessing the Val–202–Phe mutation exhibited a fitness advantage in vegetative and reproductive growth. CONCLUSION: The α-tubulin mutations Arg–243–Met and Val–202–Phe have contrasting effects on fitness. These results help understand the absence of plants homozygous for the Arg–243–Met mutation and the high frequency of plants carrying the Val–202–Phe mutation in dinitroaniline-resistant L. rigidum populations. The α-tubulin Arg–243–Met mutation can have an exceptional fitness cost with nearly lethal effects on resistant L. rigidum plants.