AbstractMidwest USA farmers have reported inconsistent control of Amaranthus tuberculatus (= rudis) (Moq ex DC) JD Sauer by glyphosate in glyphosate‐resistant crops. The potential of selection for decreased A tuberculatus sensitivity to glyphosate was therefore investigated in a reportedly resistant Everly, IA population (P0–EV). Evaluation of six A tuberculatus populations from the Midwest USA estimated a seedling baseline sensitivity of 2.15 mM glyphosate. Based on these results, three generations of divergent recurrent selection were implemented on P0–EV to isolate resistant and susceptible populations. A seedling assay was developed to screen large amounts of seeds and thus expedite the selection process. Whole‐plant and seedling rate responses of P0–EV and a known pristine A tuberculatus population from Paint Creek, OH (P0–WT) identified no significant difference in response to glyphosate; however, greater phenotypic variance was ostensibly evident in P0–EV. The first recurrent generation selected for resistance at 3.2 mM glyphosate (RS1–R) had a 5.9‐ and 1.7‐fold resistance increase at the seedling and whole‐plant levels, respectively, compared with the susceptible generation selected at 32 µM glyphosate. After three cycles of recurrent selection, 14.6‐fold difference in resistance at the seedling level and 3.1‐fold difference at the whole‐plant level were observed when comparing the populations selected for resistance (RS3–R) and susceptibility (RS3–S). Overall, recurrent selection increased the frequency of resistant individuals and decreased the variability to glyphosate at the population level. Nevertheless, variability for glyphosate resistance was still evident in RS3–R. Results herein suggested that A tuberculatus is inherently variable to glyphosate and that selection decreased the sensitivity to glyphosate. We purport that evolved glyphosate resistance in A tuberculatus may require multiple cycles of selection under field conditions. Historic estimated use of glyphosate alludes to the evolution of tolerant weed populations. Copyright © 2005 Society of Chemical Industry