Biotypes of eastern black nightshade were found in Illinois and Indiana that were not controlled by postemergence applications of imazethapyr. Greenhouse studies were conducted to determine whole-plant responses to the imidazolinone herbicides imazethapyr and imazamox and to the sulfonylurea herbicide primisulfuron-methyl. The Illinois and Indiana resistant biotypes were highly resistant to imazamox and imazethapyr but were not cross-resistant to primisulfuron-methyl. DNA sequencing of acetolactate synthase (ALS) genes showed that the molecular basis for resistance in both resistant biotypes was a single base-pair mutation within Domain C that changed an alanine to a threonine in their encoded enzymes. In vitro enzyme assays showed that the Illinois resistant biotype's ALS enzyme was 789- and 881-fold less sensitive than that of the susceptible biotype to imazamox and imazethapyr, respectively. The Indiana resistant biotype's ALS enzyme was 110- and 158-fold less sensitive than that of the susceptible biotype to imazamox and imazethapyr, respectively. These results are consistent with the amino acid substitution found in the ALS enzyme of both resistant biotypes of eastern black nightshade.