In two different locations of the Western Australian “wheatbelt”, Lolium rigidum (rigid ryegrass) seeds were collected from organic fields (no herbicide use) and neighbouring conventional fields (persistent herbicide use), the latter infested with herbicide-resistant plants, to investigate the occurrence of gene flow among field populations as revealed by herbicide resistance gene transfer. Herbicides targeting acetyl-CoA carboxylase (ACCase) or acetolactate-synthase (ALS) were used to detect herbicide-resistant plants. Overall, the frequency of plants resistant to ACCase- or ALS-inhibiting herbicides was, respectively, 21% and 74% in the conventional fields and 2% and 37% in neighbouring organic fields. Mutant, herbicideresistant ACCase and ALS alleles were detected in 16% and 38% of plants from conventional fields and in 0.53% and 3.7% of plants from organic fields. Identical mutant, herbicide-resistant ALS haplotypes were detected both in conventional and organic fields, supporting the occurrence of gene flow between L. rigidum populations in different fields. Gene flow can thus substantially increase the frequency of herbicide-resistant plants in unselected L. rigidum populations. Although gene flow cannot be prevented, it can be limited or managed. Hygiene tactics such as clean crop seed, weed seed removal at harvest and seed destruction post-harvest should be considered in order to minimize gene transfer among farms.