Two previously identified triazine-resistant annual bluegrass (Poa annua) biotypes in Mississippi were evaluated to determine the level as well as the mode of resistance compared to a triazine-susceptible biotype. Annual bluegrass seeds were collected at two sites (Meridian and Tupelo, MS) from field plots previously treated with 11.2 kg ai/ha simazine. Seeds were planted on agar containing simazine (0, 0.001, 0.01, 0.1, 1, 10, or 100 mM) or diuron (0, 0.01, 0.1, 1, 10, 100, or 1,000 μM), placed in the growth chamber with an 8 h daylength and alternating temperatures of 15 and 20 C, and allowed to grow for 3 wk, after which fresh weight was determined. Data were subjected to regression analyses and the amount of simazine or diuron required to reduce fresh weight to 50% of the untreated plants was determined. These data indicated a greater than 1,000-fold level of resistance to simazine with either biotype. Diuron concentrations of 5.0, 3.0, and 3.2 μM were required to reduce fresh weight to 50% of the untreated for the triazine-susceptible, Meridian, and Tupelo biotypes, respectively. Sequencing a portion of the chloroplast psbA gene revealed a serine 264 to glycine mutation, which reduced the affinity of the triazine herbicides for the QB-binding niche on the D1 protein in photosystem II.