In the summer of 2016, a case of potentially herbicide-resistant redroot pigweed (Amaranthus retroflexus L.). Amaranthus retroflexus was reported in Saint-Louis-de-Gonzague (Qc, Canada) in an identity preserved soybean [Glycine max (L.) Merr.] field where imazethapyr and chlorimuron-ethyl were applied pre-emergence, two active ingredients that inhibit acetolactate synthase (ALS). A few years earlier, several A. retroflexus populations resistant to ALS inhibiting herbicides were reported in Manitoba (MB). Amaranthus retroflexus samples from these fields were collected and analysed to characterize the presence of a resistance mechanism and the associated level of resistance. Sequencing of the ALS gene revealed a mutation resulting in a serine to asparagine substitution at amino acid position 653. Dose response experiments indicated resistance factors of 27.7 to 194.0 to imazethapyr (imidazolinones) among these populations, but susceptibility to members of all other ALS inhibitor families that were tested. In one MB population a serine to isoleucine substitution at position 653 was observed for the first time in A. retroflexus in some of the plants and this contributed to the detection of resistance to thifensulfuron-methyl when compared with most susceptible control population. The response of the two MB control populations to thifensulfuron-methyl differed and R factors were influenced by the level of susceptibility of the control populations to thifensulfuron-methyl. The same was not observed with imathezapyr. Non-target site resistance (NTSR) was not detected in the MB populations when exposed to malathion prior to exposure to the herbicide. We identified two new mutations on the ALS gene in A. retroflexus that result in ALS inhibitor specific cross-resistance patterns. Further investigation into NTSR and the mechanism behind the differential response of the control populations to thifensulfuron-methyl are warranted.