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Glyphosate is the most commonly used agricultural herbicide in the world. In aquatic ecosystems, glyphosate often adsorbs to benthic substrates or is metabolized and degraded by microorganisms. The effects of glyphosate on microbial communities vary widely as microorganisms respond differently to exposure. To help understand the impacts of glyphosate on the sediment microbiome, we conducted a microcosm experiment examining the responses of benthic sediment microbial communities to herbicide treatments. Sediments from a prairie pothole wetland were collected, and 16S rRNA gene sequencing was used to analyze community composition 2-h and 14-days after a single treatment of low (0.07 ppm), medium (0.7 ppm), or high (7 ppm) glyphosate, aminomethylphosphonic acid (glyphosate metabolite), or a glyphosate-based commercial formula. We found no significant differences in microbial community composition across treatments, concentration levels, or day of sampling. These findings suggest that microbial species in the Prairie Pothole Region of North America may be tolerant to glyphosate exposure., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)