ALS inhibitor-resistant biotypes are the fastest growing class of herbicide-resistant (HR) weeds. A Canadian ALS inhibitor-resistant biotype of Russian thistle was first reported in 1989. The molecular basis for ALS-inhibitor resistance is unknown for Canadian populations of this polyploid weed species, and was determined in this study for one Alberta and two Saskatchewan HR Russian thistle populations. HR plants survived spray application of the ALS-inhibitor mixture thifensulfuron : tribenuron in the greenhouse. All three HR Russian thistle populations were heterogeneous and contained both HR and herbicide-susceptible (HS) individuals. The molecular basis for resistance was determined by sequencing the ALS gene and/or conducting a TaqMan genotyping assay for single nucleotide polymorphism (SNP) for the Trp574Leu mutation. Two target-site mutations were observed: Trp574Leu in all three biotypes (554 individuals) and Pro197Gln in one biotype (one individual), suggesting multiple-founding events for Russian thistle HR populations in western Canada. Segregation patterns among F1 and F2 progeny arrays of HR lines sprayed under greenhouse conditions varied; some segregated (i.e., had HR and HS progeny), whereas other lines were exclusively HR. In contrast, no segregation of molecular types, i.e., Trp574, Trp/Leu574 and Leu574, as would be expected with heterozygosity at a single locus Trp/Leu574, was observed. Such lack of segregation is consistent with the polyploid genome structure of Russian thistle and the presence of two copies of the ALS gene. The presence of more than one ALS gene confounded the ability of the molecular techniques to accurately identify “true” heterozygotes in this study.