Multiple-herbicide resistance represents an added weed management challenge to growers as it can considerably reduce their options for weed control. The widespread nature of triazine resistance in Ontario coupled with the more recent appearance of resistance to ALS inhibitors in Amaranthus species warranted documenting biotypes with multiple resistance. A collection of Powell amaranth and redroot pigweed biotypes that had previously been characterized for resistance to ALS inhibitors was therefore screened with atrazine. Dose–response analysis with atrazine and imazethapyr was also conducted. High-level resistance to imazethapyr and atrazine was determined in a Powell amaranth biotype from Perth County, Ontario. This biotype had a > 1,860-fold and 109-fold resistance to atrazine and imazethapyr, respectively. Sequence analysis was conducted for the psbA and ALS genes that code for the target sites of the triazines and imidazolinones, respectively. A mutation in the psbA gene was identified that coded for an amino acid substitution of glycine for serine at residue 264 of the D1 protein. This mutation is the most likely cause for triazine resistance in this biotype. Similarly, a nucleotide substitution was identified that codes for threonine in place of serine at position 652 of the ALS protein. This mutation in the ALS gene has only been observed previously in laboratory-selected mutants of arabidopsis and tobacco and is known to endow resistance to imidazolinones in plants. It is concluded that multiple resistance in this Powell amaranth biotype is due to the presence of altered target sites for triazine and imidazolinone herbicides.