BACKGROUND: The pre-emergence dinitroaniline herbicides (such as trifluralin and pendimethalin) are vital to Australian no-till farming systems. A Lolium rigidum population collected from the Western Australian grain belt with a 12-year trifluralin use history was characterised for resistance to dinitroaniline, acetyl CoA carboxylase (ACCase)- and acetolactate synthase (ALS)-inhibiting herbicides. Target-site resistance mechanisms were investigated. RESULTS: This L. rigidum population exhibited 32-fold resistance to trifluralin, as compared with the susceptible population. It also displayed 12- to 30-fold cross-resistance to other dinitroaniline herbicides (pendimethalin, ethalfluralin and oryzalin). In addition, this population showed multiple resistance to commonly used post-emergence ACCase- and ALS-inhibiting herbicides. Two target-site α-tubulin gene mutations (Val-202-Phe and Thr-239-Ile) previously documented in other dinitroaniline-resistant weed species were identified, and some known target-site mutations in ACCase (Ile-1781-Leu, Asp-2078-Gly and Cys-2088-Arg) and ALS (Pro-197-Gln/Ser) were found in the same population. An agar-based Petri dish screening method was established for the rapid diagnosis of resistance to dinitroaniline herbicides. CONCLUSION: Evolution of target-site resistance to both pre- and post-emergence herbicides was confirmed in a single L. rigidum population. The α-tubulin mutations Val-202-Phe and Thr-239-Ile, documented here for the first time in L. rigidum, are likely to be responsible for dinitroaniline resistance in this population. Early detection of dinitroaniline herbicide resistance and integrated weed management strategies are needed to maintain the effectiveness of dinitroaniline herbicides.