In Argentinean crop fields, weed control is mainly achieved by intense use of glyphosate as a nonselective and/or selective herbicide. Glyphosate use is very high as more than 95% of the 16 million ha soybean crop consists of glyphosate-resistant cultivars, always treated with this herbicide. From initial success, inconsistent glyphosate control of Johnsongrass, an invading C4 perennial grass of soybean crops, has become evident to producers from northern Argentina over the last 3 yr. Prior to this, glyphosate provided good control. This study evaluated the nature of these recurrent glyphosate failures in Johnsongrass. Experiments conducted with Johnsongrass plants obtained from seed and rhizome phytomers collected from fields with intense glyphosate use history showed that these populations showed differential survival and biomass productivity when glyphosate treated than Johnsongrass plants obtained from similar propagules collected from field sites with no history of glyphosate use. This empirical evidence establishes that the Johnsongrass survival in glyphosate-treated transgenic soybean fields from northern Argentina is due to evolved glyphosate resistance.