Glyphosate-resistant (GR) crops have been adopted rapidly since their commercial introduction, and with the increase in commercially available crops resistant to glyphosate, continuous use of the same herbicide mode of action is now possible in some crop rotations. A 6-yr study was initiated to investigate the effects of conventional herbicides compared with continuous use of glyphosate in GR or Roundup Ready corn and GR soybean in a corn–soybean and a corn–soybean–winter wheat rotation. Individual experiments were fully phased and established at three locations under conventional tillage (CT) and at two locations under no-tillage (NT). Results indicated that midseason weed ground cover was lower when weeds were controlled with glyphosate; however, in most cases, this did not result in improved corn or soybean yields. Within locations, species richness, which strongly influenced other diversity indicators, was most affected by the herbicide treatments. Including winter wheat in the crop rotation had little effect on corn and soybean weed ground cover, density, and community structure and only affected soybean yield. Moreover, no effects of herbicide system used in previous corn and soybean were observed in winter wheat, with the exception of species diversity in NT, where species diversity tended to be greater when weeds in previous corn and soybean were treated with conventional herbicides. After 6 yr, the effects of continuous use of GR crops in rotation were similar to those reported in previous studies; however, continued monitoring and longer-term investigations of these systems are necessary to detect the early stages of development of herbicide-resistant biotypes.