The anticipated release of EnlistTM cotton, corn, and soybean cultivars likely will increase the use of 2,4-D, raising concerns over potential injury to susceptible cotton. An experiment was conducted at 12 locations over 2013 and 2014 to determine the impact of 2,4-D at rates simulating drift (2 g ae ha−1) and tank contamination (40 g ae ha−1) on cotton during six different growth stages. Growth stages at application included four leaf (4-lf), nine leaf (9-lf), first bloom (FB), FB + 2 wk, FB + 4 wk, and FB + 6 wk. Locations were grouped according to percent yield loss compared to the nontreated check (NTC), with group I having the least yield loss and group III having the most. Epinasty from 2,4-D was more pronounced with applications during vegetative growth stages. Importantly, yield loss did not correlate with visual symptomology, but more closely followed effects on boll number. The contamination rate at 9-lf, FB, or FB + 2 wk had the greatest effect across locations, reducing the number of bolls per plant when compared to the NTC, with no effect when applied at FB + 4 wk or later. A reduction of boll number was not detectable with the drift rate except in group III when applied at the FB stage. Yield was influenced by 2,4-D rate and stage of cotton growth. Over all locations, loss in yield of greater than 20% occurred at 5 of 12 locations when the drift rate was applied between 4-lf and FB + 2 wk (highest impact at FB). For the contamination rate, yield loss was observed at all 12 locations; averaged over these locations yield loss ranged from 7 to 66% across all growth stages. Results suggest the greatest yield impact from 2,4-D occurs between 9-lf and FB + 2 wk, and the level of impact is influenced by 2,4-D rate, crop growth stage, and environmental conditions.