Annual bluegrass is the most problematic winter annual weed in managed turfgrass. Acetolactate synthase (ALS)-inhibiting herbicides are effective for annual bluegrass control, but reliance on this mode of action can select for herbicide-resistant biotypes. Two annual bluegrass biotypes not controlled with ALS-inhibiting herbicides were reported at golf courses in South Carolina and Georgia. Research was initiated at Clemson University to verify the level of resistance of these biotypes to ALS inhibitors. Two ALS-susceptible (S) and suspected resistant (SCr, GAr) annual bluegrass biotypes were established in a greenhouse. Dose-response experiments were conducted on mature annual bluegrass plants using trifloxysulfuron, foramsulfuron, and bispyribac-sodium, all ALS-inhibiting herbicides. Additionally, a rapid diagnostic ALS activity assay was optimized and conducted using the same herbicides. For dose-response experiments, the rate of herbicide that reduced shoot biomass 50% (I50) values for the S biotypes were 13.6 g ai ha−1 for trifloxysulfuron, 7.0 g ai ha−1 for foramsulfuron, and 38.3 g ai ha−1 for bispyribac-sodium. Fifty percent shoot biomass reduction was not observed in either the SCr or GAr biotypes at eight times the labeled field rate of all ALS-inhibiting herbicides tested. For in vivo tests of ALS activity, the SCr biotype yielded I50 (concentration of herbicide that reduced ALS activity 50%) values 3,650, 3,290, and 13 times the S biotypes following treatment with trifloxysulfuron, foramsulfuron, and bispyribac-sodium, respectively. Similarly, I50 values for the GAr biotype were 316, 140, and 64 times greater than the S biotypes following the same herbicide treatments. This research indicates high levels of annual bluegrass resistance to multiple ALS-inhibiting herbicides in South Carolina and Georgia. Future research should focus on the mechanisms of ALS resistance in these annual bluegrass biotypes as well as alternative options for control not targeting the ALS enzyme.