Methiozolin is a new herbicide from South Korea currently under development in the United States for PRE and POST annual bluegrass control in bentgrass and most other cool- and warm-season turfgrasses. Greenhouse studies were conducted in 2012 at the University of California, Riverside, CA, and Auburn University, Auburn, AL, to evaluate the relative tolerance of three bentgrass species comprised of nine creeping bentgrass (CRBG) cultivars, velvet bentgrass (VBG) and colonial bentgrass (COBG) to methiozolin at 0, 0.6, 1.1, 2.2, 4.5 and 9.0 kg ai ha−1. Methiozolin was applied 7 wk after seeding, followed by a second application 5 wk later. Methiozolin rates that produced 25% injury (TI25) and 50% clipping dry weight reduction (GR50) relative to a nontreated control for each species or cultivar were calculated using four-parameter logistic regression. Turf injury rates at 21 d after second treatment (DAT2) were the most consistent in describing relative tolerance among bentgrass species. Overall, CRBG was more tolerant to methiozolin than VBG or COBG. After two applications, methiozolin rates that caused TI25 were 1.1, 0.2, and 0.3 kg ha−1 for CRBG (across all cultivars), VBG, and COBG, respectively. VBG and COBG were not tolerant of sequential methiozolin applications at rates necessary to control annual bluegrass under field conditions. Herbicide rates that caused TI25 and GR50 decreased with the second application. ‘Penn A-4’ CRBG exhibited the highest TI25 28 d after initial treatment (DAIT) at University of California at Riverside (4.5 kg ha−1), but only 2.5 kg ha−1 with two applications by 21 DAT2. All CRBG cultivars tested tolerated methiozolin at 0.5 kg ha−1, the recommended sequential use rate for putting greens in Korea.