Annual bluegrass is commonly controlled by acetolactate synthase (ALS)-inhibiting herbicides in managed turfgrass. An annual bluegrass population with suspected resistance to ALS-inhibiting herbicides was collected from Grand National Golf Course in Opelika, AL (GN population). Subsequent testing confirmed resistance of the GN population to foramsulfuron, trifloxysulfuron, bispyribac-sodium (bispyribac), and imazaquin when compared to a susceptible population collected locally at Auburn University (AU population). Sequencing of the ALS gene revealed a point mutation resulting in an amino acid substitution at Trp574. Cloning of the ALS gene surrounding the Trp574 region yielded two distinct ALS gene sequences: one producing Trp574 and one producing Leu574. Trp574 to Leu has been previously correlated with resistance to ALS-inhibiting herbicides. Both AU and GN gene sequences contained other similar silent and missense mutations. This research confirms resistance of annual bluegrass to ALS-inhibiting herbicides with Trp574 to Leu amino acid substitution being the most likely mode of resistance based on past literature.