Over 90% of Canadian kochia populations are resistant to acetolactate synthase (ALS)– inhibiting herbicides. We questioned whether the target site–based resistance could affect plant growth and competitiveness. Homozygous F2 herbicide-resistant (HR) kochia plants with an amino acid substitution at Trp574 (sources: Alberta [AB], Saskatchewan [SK], and Manitoba [MB]), or Pro197 (MB, AB with two populations) were grown in replacement series with homozygous F2 herbicide-susceptible (HS) plants from the corresponding heterogeneous population (total: six populations). In pure stands, growth of HR plants from AB and SK was similar to that of HS plants, regardless of mutation; conversely, MB2-HR plants (Trp574Leu) developed more slowly and were taller than MB2-HS plants. Final dry weight of HR plants in pure stands was similar across all six populations, whereas that for HS plants in pure stands and HR–HS plants in mixed stands (50–50%) varied with population. Results for AB and SK populations suggest little impact of either ALS mutation on kochia growth, whereas those for MB lines would suggest an unidentified factor (or factors) affecting the HS, HR, or both biotypes. The variable response within and between lines, and across HS biotypes highlights the importance of including populations of various origins and multiple susceptible controls in HR biotype studies.