Soybean is a major crop cultivated in Brazil, and acetolactate synthase (ALS)-inhibiting herbicides are widely used to control weeds in this crop. The continuous use of these ALS-inhibiting herbicides has led to the evolution of herbicide-resistant weeds worldwide. Greater beggarticks is a polyploid species and one of the most troublesome weeds in soybean production since the discovery of ALS-resistant biotypes in 1996. To confirm and characterize the resistance of greater beggarticks to ALS inhibitors, whole-plant bioassays and enzyme experiments were conducted. To investigate the molecular basis of resistance in greater beggarticks the ALS gene was sequenced and compared between susceptible and resistant biotypes. Our results confirmed that greater beggarticks is resistant to ALS inhibitors and also indicated it possesses at least three isoforms of the ALS gene. Analysis of the nucleotide and deduced amino acid sequences among the isoforms and between the biotypes indicated that a single point mutation, G–T, in one ALS isoform from the resistant biotype resulted in an amino acid substitution, Trp574Leu. Two additional substitutions were observed, Phe116Leu and Phe149Ser, in a second isoform of the resistant biotype, which were not yet reported in any other herbicide-resistant ALS gene; thus, their role in conferring herbicide resistance is not yet ascertained. This is the first report of ALS mutations in an important, herbicide-resistant weed species from Brazil.