aims: Glyphosate-resistant (GR) soybean production increases each yearbecause of the efficacy of glyphosate for weed management. A new or ‘second’generation of GR soybean (GR2) is now commercially available for farmersthat is being promoted as higher yielding relative to the previous, ‘first genera-tion’ (GR1) cultivars. Recent reports show that glyphosate affects the biologyand ecology of rhizosphere micro-organisms in GR soybean that affect yield.The objective of this research was to evaluate the microbiological interactionsin the rhizospheres of GR2 and GR1 soybean and the performance of the culti-vars with different rates of glyphosate applied at different growth stages.Methods and Results: A greenhouse study was conducted using GR1 and GR2soybean cultivars grown in a silt loam soil. Glyphosate was applied at V2, V4and V6 growth stages at three rates. Plants harvested at R1 growth stage hadhigh root colonization by Fusarium spp.; reduced rhizosphere fluorescent pseu-domonads, Mn-reducing bacteria, and indoleacetic acid–producing rhizobacte-ria; and reduced shoot and root biomass.Conclusions: Glyphosate applied to GR soybean, regardless of cultivar, nega-tively impacts the complex interactions of microbial groups, biochemical activ-ity and root growth that can have subsequent detrimental effects on plantgrowth and productivity.Significance and Impact of the Study: The information presented here will becrucial in developing strategies to overcome the potential detrimental effects ofglyphosate in GR cropping systems.