Abstract Amaranthus retroflexus L. is a troublesome broadleaf weed in autumn crop fields in China. Farmers complain that A. retroflexus cannot be controlled by nicosulfuron at the recommended field rate in maize fields in Heilongjiang Province. The aim of this study was to determine the molecular basis of nicosulfuron resistance in A. retroflexus. Whole-plant response assays revealed that two resistant populations (R1 and R2) exhibited resistance (14.50- and 44.24-fold) to nicosulfuron. In vitro acetolactate synthase (ALS) activity assays indicated that the nicosulfuron I50 values for R1 and R2 populations were 8.12 and 22.32 times higher than that for the susceptible population (S), respectively. Sequence analysis of ALS showed amino acid mutations Ser-653-Asn in R1 and Trp-574-Leu in R2. These two target-site mutations seem to play a primary role in A. retroflexus resistance to nicosulfuron, and this is the first time that the Ser-653-Asn mutation has been reported in this species.