A segment of the debate surrounding the commercialization of genetically engineered (GE) crops, such as glyphosate-resistant (GR) crops, focuses on the theory that implementation of these traits is an extension of the intensification of agriculture that will further erode the biodiversity of agricultural landscapes. A large field-scale study was conducted in 2006 in the United States on 156 different field sites with a minimum 3-yr history of GR corn, cotton, or soybean in the cropping system. The impact of cropping system, crop rotation, frequency of using the GR crop trait, and several categorical variables on emerged weed density and diversity was analyzed. Species richness, evenness, Shannon's H′, proportion of forbs, erect growth habit, and C3 species diversity were all greater in agricultural sites that lacked crop rotation or were in a continuous GR crop system. Rotating between two GR crops (e.g., corn and soybean) or rotating to a non-GR crop resulted in less weed diversity than a continuous GR crop. The composition of the weed flora was more strongly related to location (geography) than any other parameter. The diversity of weed flora in agricultural sites with a history of GR crop production can be influenced by several factors relating to the specific method in which the GR trait is integrated (cropping system, crop rotation, GR trait rotation), the specific weed species, and the geographical location. The finding that fields with continuous GR crops demonstrated greater weed diversity is contrary to arguments opposing the use of GE crops. These results justify further research to clarify the complexities of crops grown with herbicide-resistance traits, or more broadly, GE crops, to provide a more complete characterization of their culture and local adaptation.