Annual bluegrass is a pervasive weed on golf courses in the Transition Zone of the United States and is difficult to selectively remove. For years, superintendents have applied glyphosate on dormant zoysiagrass to remove cool-season weeds. In 2007, a population of annual bluegrass in Columbia, MO, was not controlled with glyphosate after more than 10 yr of continuous applications. Greenhouse studies were established to compare the response of suspect glyphosate-resistant (CCMO1) and -susceptible annual bluegrass to glyphosate. Seedling plants were treated with glyphosate from 0 to 6.27 kg ae ha−1. At 21 d after treatment, reductions in biomass for susceptible annual bluegrass reached a maximum at glyphosate rates of 0.78 kg ha−1 or higher. Comparatively, the biomass of CCMO1 plants was only reduced by 50% at 0.78 kg ha−1, and reductions did not exceed 60% at rates up to 6.27 kg ha−1, which is eight times the labeled rate. At rates necessary to reduce plant dry weights by 50%, the resistance factor (RF) for CCMO1 was 5.2. Twenty-one days following biomass assessment, regrowth of plants was non-existent on susceptible plants at 0.78 kg ha−1 glyphosate or above, but CCMO1 plants reached 1.7 cm regrowth at the 6.27 kg ha−1 rate. Based on the regrowth, the RF for CCMO1 was 5.2. Results indicate a new species has been identified with resistance to glyphosate, and this represents the first report of glyphosate resistance in turfgrass.