BackgroundA landscape‐scale probability‐based sampling of Iowa soybean [Glycine max (L.) Merr.] fields was conducted in 2013 and 2019; Amaranthus tuberculatus [Moq.] J.D. Sauer seed was collected from 97 random geospatial selected fields. The objectives were to evaluate the prevalence and distribution of herbicide‐resistant A. tuberculatus (waterhemp) in soybean fields and evaluate temporal changes over 6 years. Amaranthus tuberculatus seedlings were evaluated for resistance to imazethapyr, atrazine, glyphosate, lactofen and mesotrione at 1× and 4× label rates.ResultsResistance to imazethapyr, glyphosate, lactofen and mesotrione at the 1× rate increased significantly from 2013 to 2019 and was found in 99%, 97%, 16% and 15% of Iowa A. tuberculatus populations in 2019, respectively. Resistance to atrazine at the 4× rate increased over time; atrazine resistance was found in 68% of populations in 2019. Three‐way multiple herbicide‐resistant A. tuberculatus was the most frequent and increased significantly to 4× rates from 16% in 2013 to 43% of populations in 2019. All A. tuberculatus populations resistant to HPPD‐inhibitor herbicides also were resistant to atrazine.ConclusionTo the best of our knowledge, this is the first probability‐based study that presented evolution of A. tuberculatus herbicide resistance over time. The results demonstrated that imazethapyr, atrazine and glyphosate resistance in Iowa A. tuberculatus populations was frequent whereas resistance to lactofen and mesotrione was less frequent. Most Iowa A. tuberculatus populations evolved resistance to multiple sites of action over time. The results of our study are widely applicable given the similarities in weed management practices throughout the Midwest United States. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.