Johnsongrass is a common weed of corn in Chile, which is most often controlled by nicosulfuron, an acetohydroxyacid synthase (AHAS)-inhibiting herbicide. Recurrent nicosulfuron use has resulted in selection for resistant johnsongrass biotypes. We conducted studies to determine nicosulfuron resistance levels in two johnsongrass biotypes from Chile and to investigate if this resistance was target-site mediated. Whole-plant resistance to nicosulfuron was 33 and 46 times higher in resistant (R) than in susceptible (S) plants grown from seed and rhizomes, respectively. The nicosulfuron concentrations for 50% inhibition of AHAS enzyme activity in vitro were more than 11 times higher in R than in S plants. Sequencing analysis of the AHAS coding sequence revealed a Trp-574-Leu substitution in both R biotypes. This study shows that resistance to nicosulfuron in the two R biotypes is conferred by an altered target site. We also report the first consensus sequence of the johnsongrass AHAS gene corresponding to the known mutation sites conferring resistance to AHAS-inhibiting herbicides.