Abstract The composition and diversity of the bacterial community associated with plant roots is influenced by a variety of plant factors such as root density and exudation. In turn, these factors are influenced by plant breeding programs. This study assessed the diversity of root-endophytic and rhizosphere bacterial communities associated with three canola cultivars (Parkland, Brassica rapa; Excel, B. napus; and Quest, B. napus) grown at two field sites. Quest, a derivative of Excel developed by the Alberta Wheat Pool, has been genetically engineered to tolerate the herbicide glyphosate. Approximately 2300 bacteria were isolated from roots of plants and identified based on fatty acid methyl ester (FAME) profiles. One third of the isolates were positively identified by FAME analysis (i.e. SIM index ≥0.3) with another third assigned tentative identifications (SIM index <0.3). Fewer Bacillus, Micrococcus and Variovorax isolates, and more Flavobacterium and Pseudomonas isolates were found in the root interior of Quest compared to Excel or Parkland. Furthermore, fewer Arthrobacter and Bacillus isolates were recovered from the rhizosphere of Quest compared to Excel or Parkland. The bacterial root-endophytic community of the transgenic cultivar, Quest, was separated by principal component analysis from the other cultivars, and exhibited a lower diversity compared to Excel or Parkland. The rhizosphere of all cultivars yielded more Arthrobacter, Aureobacterium, and Bacillus isolates, but fewer Micrococcus, Variovorax and Xanthomonas isolates compared to the root interior. The results from this study indicate that the composition of the root-endophytic bacterial community of canola differs between cultivars.