Resistance to acetohydroxyacid synthase (AHAS) inhibiting herbicides in populations of eastern black nightshade from Ontario has been linked to an Ala205Val substitution in the AHAS enzyme. The aim of this study was to determine the mechanism of inheritance of AHAS inhibitor resistance and the genetic relationships among resistant (R) and susceptible (S) eastern black nightshade populations from Ontario. Homozygous R and S parental populations were crossed and the inheritance was analyzed in F1 (S × R), reciprocal F1 (R × S), F2, and backcross (S × F1) progenies after application of imazethapyr at 150 g ai ha−1. Compared to parental lines, the progenies were rated as R, intermediate (I), and S phenotypes. All the F1 progenies were of the I phenotype. The backcross progenies segregated in a 1:1 (S:I) ratio, and the F2 families segregated in a 1:2:1 (R:I:S) ratio. These results indicate that a single nuclear gene, with incomplete dominance, controls resistance to AHAS-inhibiting herbicides in R population of eastern black nightshade. Random amplified polymorphic DNA (RAPD) markers were screened among 25 R and S populations. The genetic relationship of R and S populations based on RAPD profiles generated from six RAPD primers indicated four groups of populations in which resistance seems to have arisen independently. However, based on similarity coefficients, resistance within three of the groups could have arisen by gene flow. Both similar local selection pressure and gene flow could explain the spread of the Ala205Val substitution in R populations of eastern black nightshade in Ontario.