Acetolactate synthase (ALS), a common enzyme in the biosynthesis of branched-chain amino acids, is a target for the sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidinylthiobenzoate, and the sulfonylaminocarbonyltriazolinone classes of herbicides. Widespread resistance to the ALS-inhibiting herbicides has been attributed to single-base mutations in the ALS gene. The objective of this study was to investigate the feasibility of using denaturing high-performance liquid chromatography (DHPLC), a recently developed method of mutation analysis, for the detection of three ALS mutations, Ala122Thr, Leu574Trp, and Ser653Thr, which confer herbicide resistance. The mutated variants of the ALS gene were isolated from herbicide-resistant biotypes of smooth pigweed and Powell amaranth using polymerase chain reaction (PCR). The PCR products were hybridized with a wild-type sample and subjected to DHPLC analysis. All three mutations could be detected using a modified High performance liquid chromatography system; however, the sensitivity of the method was strongly dependent on the melting temperature profile of the analyzed DNA fragment. Once the primers and the DHPLC conditions are optimized, the procedure is economical, rapid, and requires little sample preparation. Because of these favorable features, DHPLC can be used as an alternative to other commonly used mutation detection methods.