Methiozolin is a new isoxazoline herbicide being investigated for selective POST annual bluegrass control in creeping bentgrass putting greens. Glasshouse and field research was conducted from 2010 to 2012 in Tennessee and Texas to evaluate annual bluegrass control efficacy with methiozolin. Application placement experiments in the glasshouse illustrated that root absorption was required for POST annual bluegrass control with methiozolin at 1,000 g ai ha−1. Soil-plus-foliar and soil-only applications of methiozolin reduced annual bluegrass biomass greater than treatments applied foliar-only. Field experiments evaluated annual bluegrass control efficacy with two application rates (500 and 1,000 g ha−1) and six application regimes (October, November, December, October followed by [fb] November, November fb December, and October fb November fb December) on sand- and soil-based putting greens. Annual bluegrass control with methiozolin at 1,000 g ha−1 on sand-based greens ranged from 70 to 72% compared to 87 to 89% on soil-based greens. Treatment at 500 g ha−1 controlled annual bluegrass 57 to 64% on sand-based greens compared to 72 to 80% on soil-based greens. Most sequential methiozolin application regimes controlled annual bluegrass more than single applications. On sand-based greens, sequential application programs controlled annual bluegrass 70 to 79% compared to 85 to 92% on soil-based greens. Responses indicate that methiozolin is a root-absorbed herbicide with efficacy for selective control of annual bluegrass in both sand- and soil-based creeping bentgrass putting greens.