ALS inhibitor-resistant biotypes are the fastest growing class ofherbicide-resistant (HR) weeds. A Canadian ALS inhibitor-resistant biotypeof Russian thistle was first reported in 1989. The molecular basis forALS-inhibitor resistance is unknown for Canadian populations of thispolyploid weed species, and was determined in this study for one Alberta andtwo Saskatchewan HR Russian thistle populations. HR plants survived sprayapplication of the ALS-inhibitor mixture thifensulfuron : tribenuron in thegreenhouse. All three HR Russian thistle populations were heterogeneous andcontained both HR and herbicide-susceptible (HS) individuals. The molecularbasis for resistance was determined by sequencing the ALSgene and/or conducting a TaqMan genotyping assay for single nucleotidepolymorphism (SNP) for the Trp574Leu mutation. Two target-sitemutations were observed: Trp574Leu in all three biotypes (554individuals) and Pro197Gln in one biotype (one individual),suggesting multiple-founding events for Russian thistle HR populations inwestern Canada. Segregation patterns among F1 and F2 progeny arrays of HRlines sprayed under greenhouse conditions varied; some segregated (i.e., hadHR and HS progeny), whereas other lines were exclusively HR. In contrast, nosegregation of molecular types, i.e., Trp574, Trp/Leu574 and Leu574, as would be expected withheterozygosity at a single locus Trp/Leu574, was observed. Suchlack of segregation is consistent with the polyploid genome structure ofRussian thistle and the presence of two copies of the ALSgene. The presence of more than one ALS gene confounded theability of the molecular techniques to accurately identify “true”heterozygotes in this study.