AbstractWhile surveying Illinois Amaranthus tuberculatus (Moq) Sauer (tall waterhemp) half‐sib populations for herbicide response variability, several were observed to segregate for resistance to atrazine. Studies were conducted on greenhouse‐grown A tuberculatus plants to compare atrazine responses among populations that were segregating for resistance (SegR), uniformly sensitive (UniS) or uniformly resistant (UniR). In chlorophyll fluorescence assays, leaves of plants from the SegR and UniS populations displayed changes in fluorescence after treatment with atrazine, indicating that atrazine was inhibiting electron transport of photosystem II in chloroplasts. Sequencing of a fragment of psbA, which encodes the D1 protein, revealed that the SegR population did not contain the amino acid substitution that is typically found in triazine‐resistant plants. Whole‐plant herbicide dose‐response experiments revealed that, relative to the UniS population, atrazine resistances in the UniR and SegR populations were >770‐fold and 16‐fold, respectively. The SegR population was also resistant to cyanazine (59‐fold), but not to metribuzin, linuron or pyridate. Triazine resistance in the SegR population was shown to be a nuclear inherited trait, unlike maternal inheritance of site‐of‐action mediated triazine resistance found in the UniR population. Taken collectively, these findings confirm the existence of two distinct triazine resistance mechanisms in A tuberculatus. Copyright © 2003 Society of Chemical Industry