Evaluations of cotton exposed to 2,4-D at various growth stages were conducted at 12 locations across the cotton belt during 2013 and 2014. Two sub-lethal rates of 2,4-D, representing a physical drift scenario (1/421 of the full rate) and a tank contamination scenario (1/21 of the full rate), were applied. Applications were made at six different growth stages; four leaf (4-lf), 9 leaf (9-lf), first bloom (FB), FB+2wk, FB+4wk, and FB+6wk. Locations were grouped according to the severity of yield loss compared to non-treated cotton, resulting in three groups each containing four locations. Group I consisted of locations in which mild yield loss (≤ 15%) occurred, Group II consisted of locations which experienced a moderate yield loss (16 to 25%), while Group III consisted of locations that experienced the most severe yield loss (26 to 40%). Across the groups, the tank contamination rate was responsible for the majority (74%) of instances in which significant yield loss occurred. When significant, the drift rate of 2,4-D resulted in yield loss ranging from 19 – 40% compared to non-treated yield. The contamination rate resulted in yield loss ranging from 16 – 81% compared to non-treated yield. The greatest magnitude of yield loss occurred when 2,4-D was applied at FB, followed by applications at FB+2 wk and 9-lf. Visual injury symptoms were evaluated at eight of the locations, however, they did not reflect the severity of yield loss resulting from 2,4-D applications near FB due to the lack of vegetative injury symptoms present. Plant mapping measurements determined that when significant yield loss occurred, it was primarily a result of a reduction in the number of bolls present on the plant at harvest. This study suggests that 2,4-D would have the most severe effect on yield when cotton is exposed during growth stages near FB.