The yield response of cotton, Gossypium hirsulum L., to twospotted spider mite, Tetranychus urticae Koch, duration of infestation during the seedling stage was measured at Starkville and Stoneville, MS, during 2010 and 2011. The treatments included a noninfested control, infestations lasting for 7, 14, 21, or 28 d, and a season long infested control. Twospotted spider mites from a greenhouse colony were inoculated on all of the infested cotton plots during the three-leaf stage. Applications of miticides were made to terminate infestations at the desired timings for each treatment. Twospotted spider mite densities and injury ratings were determined for each treatment at the end of the infestation period and yield was measured at the end of the season. Twospotted spider mite densities and injury rating significantly increased as duration of infestation increased. Significant differences in yield were observed between treatments. These data suggest infestations initiated at the three-leaf cotton growth stage with population densities at a minimum of 0.39 mites/cm² existing >14 d could result in reduction of cotton yields.