Plant roots significantly affect microbial diversity in soil, but little is known on how genetically modified plants influence soil microbial communities. We conducted a 2-year field study to assess the effects of herbicide-tolerant genetically modified canola (oilseed rape, Brassica sp.) on microbial biodiversity in the rhizosphere. During the 1998 and 1999 field seasons, four genetically modified and four conventional canola varieties were grown at four different field locations across Saskatchewan, Canada. The rhizosphere and root interior microbial communities were characterized through fatty acid methyl ester analysis and community level physiological profiles. Principal component analysis indicated that the root interior and rhizosphere bacterial community associated with the genetically modified variety Quest (Brassica napus) was different from conventional varieties Excel (B. napus) and Fairview (Brassica rapa), based on both fatty acid composition and carbon substrate utilization. In addition, all root-associated microbial communities associated with genetically modified canola varieties had significantly higher levels of 10:02OH, 12:02OH, 12:03OH, a15:0, 15:1ω5c, cy17:0, 18:3ω6,9,12c, 19:0ω8c and Sum in Feature 3, suggesting alterations in the composition of the microbial community associated with plants. This study indicates that the composition and functional diversity and the microbial community were influenced by plant variety.