BACKGROUND: Indaziflam is an alkylazine herbicide used to control annual bluegrass (Poa annua L.). Several locations in the southeastern USA reported poor annual bluegrass control following treatment with indaziflam during autumn 2015. A series of controlled environment experiments were conducted to confirm putative resistance to indaziflam in annual bluegrass collected from these field locations.RESULTS: Indaziflam (25 g ha −1 ) effectively controlled all putative-resistant annual bluegrass collections when applied preemergence (PRE), but was ineffective when applied early-postemergence (< 2.5 cm plant height; BBCH scale = 1; EPOST). In agarose-based plate assays, EPOST I 50 values for putative-resistant collections ranged from 2424 to 4305 pM compared with 633 pM for the herbicide-susceptible control; therefore, resistance indexes (R/S) ranged from 3.8 to 6.8. Resistant collections were not controlled by foramsulfuron, flumioxazin, glyphosate, glufosinate, metribuzin, pronamide, or simazine applied EPOST. Indaziflam content in herbicide-susceptible annual bluegrass was greater than a resistant collection from 0 to 10 days after treatment (DAT). Susceptibility was not restored when resistant collections were treated with indaziflam plus 1-aminobenzotriazole (10 mg L −1 ), tebuconazole (1510 g ha −1 ), or malathion (400 g ha −1 ). CONCLUSIONS: This is a first report of resistance to indaziflam in any plant. Additionally, we confirm that these annual bluegrass collections are resistant to several other herbicidal modes-of-action. It is unclear if this multi-herbicide resistance is due to a single resistance gene, multiple resistance genes, non-target site mechanisms, or a combination thereof. Additional research to better understand resistance mechanisms in these annual bluegrass collections is warranted.