Horseweed can be a problematic weed in no-till soybean fields and populations can vary in their response to 2,4-D. The objective of this study was to evaluate the growth and seed production of four horseweed populations after exposure to 2,4-D. 2,4-D amine was applied at 0, 140, 280, and 560 g ae ha−1 to 5- to 10-cm-tall horseweed plants. An additional treatment of 280 g ha−1 of 2,4-D + 840 g ae ha−1 of glyphosate was included in the study. At 2 wk after treatment (WAT), injury ranged from 47 to 98%, but by 6 WAT the injury ranged from 89 to 100% for all four populations. Between 6 and 12 WAT some individual horseweed plants started to recover. No differences in dry weights were observed between the four populations in the untreated checks at 0, 2, 6, and 12 WAT. At 280 g ha−1 of 2,4-D amine, seed production was reduced by greater than 95%. However, three of the four horseweed populations had plants that survived and produced seed after exposure to 840 g ha−1 of glyphosate + 280 g ha−1 of 2,4-D. One plant produced seed after exposure to 560 g ha−1 of 2,4-D. These results suggest that horseweed can evolve resistance to 2,4-D and no fitness penalities were observed in populations that had higher levels of tolerance to 2,4-D.