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The widespread use of glyphosate-resistant (GR) cropping systems may impact rhizosphere microbial associations and crop productivity. It was previously reported that glyphosate accumulation in the rhizosphere may stimulate colonization of soybean [Glycine max (L.) Merr.] roots by soilborne Fusarium. Field studies often reveal inconsistent root colonization by Fusarium, especially during growing seasons characterized by contrasting rainfall patterns. Therefore, this study was conducted to determine the impact of different soil moisture contents on root colonization of glyphosate-treated soybean by Fusarium species. Glyphosate (0.84 kg ae ha-1) was applied to greenhouse-grown glyphosate-resistant (GR) soybean at the two to three trifoliate-leaf (V2–V3) growth stage growing in a Mexico silt loam at 27%, 13%, and 10% soil moisture contents. Soil and plant samples were sampled periodically after herbicide application and selectively cultured for Fusarium. Highest Fusarium colonization was associated with the glyphosate treatment, with maximum levels occurring at the highest soil moisture level. Thus, glyphosate interactions with root colonization by Fusarium in glyphosate-resistant soybean are greatly influenced by soil moisture content.