Resistance to acetohydroxyacid synthase (AHAS) inhibitors in populations of eastern black nightshade has been rising rapidly in Ontario. The objectives of this study were to (i) identify the spectrums of resistance to different AHAS inhibiting herbicides, (ii) identify effective alternative mode-of-action herbicides, (iii) examine how the resistant (R) AHAS behaves in response to different herbicide and end-product concentrations, (iv) determine the molecular basis of resistance, (v) determine the inheritance of resistance and the genetic variability among geographically distinct populations, and (vi) ascertain whether the resistant populations suffered from a fitness penalty under various light, watering, temperature, and intraspecific competition conditions. Compared to a susceptible (S) population one of the R populations had 726-, 31-, 6-, and 4-fold resistance to imazethapyr, imazamox, primisulfuron, and flumetsulam, respectively. There was no cross resistance in those populations to chloroacetamide herbicides. These results were confirmed with in vitro enzyme assays. Moreover, the resistant enzyme had lower activity and was less sensitive to feedback inhibition from branched-chain amino acids compared to the susceptible enzyme. The results of 'AHAS' sequencing revealed that resistance in twelve populations was due to an Ala205Val substitution, while in one population it was due to Ala122Thr substitution. A single nuclear gene, with incomplete dominance, controlled resistance to AHAS-inhibiting herbicides in R populations of eastern black nightshade. Random Amplified Polymorphic DNA (RAPD) analysis identified four distinct groups of populations, suggesting that resistance could have occurred as a result of both local selection pressure and gene flow. Fitness experiments under various light, watering, and temperature regimes, as well as competition studies indicated that under optimal reproductive conditions, Ala205Val is associated with a fitness cost in plants. There was no impact on above ground biomass production but there was a reduction in berry production and a delay in the maturation of berries. The higher concentration of branched-chain amino acids could be the reason for the lower fitness of the R populations, by diverting the resources away from the reproductive function of the mutant plants. However, the Ala205Val substitution could have modified the wild type phenotypes to adapt to stressful environments.