Three putative resistant Amaranthus retroflexus L. populations were collected in Heilongjiang province in China. Whole plant bioassays indicated high resistance (RI > 10) to imazethapyr in the three populations. In vitro acetolactate synthase (ALS) assays revealed that ALS from populations H3, H17 and H39 was less sensitive to imazethapyr inhibition compared to the susceptible population H76. The half-maximal inhibitory concentration (I50) values for H3, H17 and H39 were 14.83, 15.27 and 268 times greater, respectively, than that of the susceptible population H76. Three nucleotide mutations resulted in three known resistance-endowing amino acid substitutions, Ala-205-Val, Trp-574-Leu and Ser-653-Thr in the three resistant populations respectively. Therefore, ALS target-site mutations in resistant A. retroflexus could be responsible for imazethapyr resistance.