Weed free field experiments were conducted for 3 yr at one location to measure the response of cotton to glyphosate spot applied once, twice, and three times. Glyphosate treatments frequently used for silverleaf nightshade control were applied at specified intervals after cotton emergence to in-row, uniformly spaced densities of “simulated” weeds. The number and application timing influenced cotton injury each year. Frequently, cotton lint yields following treatments applied once at four, six, or eight sites/9 m of row were not reduced significantly compared to the untreated plots; however, average yield reductions ranged from 10 to 14%. Glyphosate, applied more than once generally, caused more crop injury and reduced lint yields by 13 to 39%.