Abstract: To investigate the molecular basis of resistance to nicosulfuron in Amaranthus retroflexus L., the whole-plant dose-response assays were conducted to determine the level of resistance. And the in vitro acetolactate synthase (ALS) inhibition assay and ALS gene sequence analysis were conducted to clarify the target-site based resistance mechanism. Results of whole-plant dose-response experiments showed that the resistant population (HLJ-R) had evolved high-level (13.7-fold) of resistance to nicosulfuron. The in vitro ALS activity assay showed that the IC₅₀ value of nicosulfuron in the resistant population (HLJ-R) was 43.9-folds higher than that of the sensitive population (TA-S). The ALS gene sequence analysis showed that two ALS resistance mutation occurred in the resistant plants, including the alanine acid (Ala) at the position 205 substituted by valine acid (Val), and tryptophan acid (Trp) at the position 574 substituted by leucine acid (Leu). These findings indicated that the reduced ALS sensitivity to nicosulfuron resulted from Ala-205-Val and Trp-574-Leu, which was the molecular basis for the target-site based resistance to nicosulfuron in A. retroflexus.