Field trials were conducted in 2001 at the Tobacco Research Station near Oxford, NC, and in 2002 at the Lower Coastal Plains Research Station near Kinston, NC, to determine tobacco yield, injury, and shikimic acid accumulation in response to simulated glyphosate drift. Glyphosate was applied to 12- to 13-cm-high tobacco ‘K326’ early postemergence at 0, 9, 18, 35, 70, 140, 280, 560, and 1,120 (1×) g ai/ha. Crop injury was rated 7 and 35 d after treatment (DAT) and shikimic acid accumulation in leaves at 7 DAT, tobacco yield, and leaf grade index (whole-plant index of harvest interval leaf value) were also assessed. Shikimic acid accumulation and injury symptoms increased similarly as glyphosate rate increased. Glyphosate rates of 140 g/ha (0.125 of recommended rate) or higher resulted in significant crop injury, reduced tobacco yield, and decreased leaf grade index. Shikimic acid accumulation at 7 DAT was inversely related to tobacco yield. Shikimic acid accumulation was found to be an effective diagnostic tool to determine glyphosate drift in tobacco; however, in-season data are needed to correlate shikimic acid accumulation with yield loss.