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Herbicide-resistant weeds are a constraint to weed management in many cropping regions around the world. Littleseed canarygrass (Phalaris minor Retz.), the most predominant weed of wheat crop has developed resistance against isoproturon in North-Western Indo-Gangetic plains of India. The herbicide-binding region of the chloroplast psbA gene from a total of eight biotypes of P. minor resistant and susceptible to isoproturon was selectively amplified using PCR. Sequence analysis of the fragment from five herbicide-resistant biotypes and three sensitive biotypes exhibited none of the six substitutions as reported in other weed species of the D1 protein encoded by the psbA gene. The lack of mutations in herbicide binding region of psbA gene of isoprotutron resistant biotypes indicated that target-site resistance mechanism does not account for resistance rather resistance to this herbicide can be attributed to non target-site resistance mechanism.