Plant characteristics are known to alter endophytic and rhizosphere microbial communities; however, the effect of crop breeding programs on the microbial endophytic and rhizosphere communities is not clear. The purpose of this study was to determine if root-associated microbial communities differed between three cultivars of canola (Brassica spp.) and wheat (Triticum spp.). BiologTM analysis was used to characterize the microbial communities associated with the root interior and rhizosphere soil of field grown canola (Parkland, Excel, and Quest cultivars) as well as wheat (PI 167549, Red Fife, and CDC Teal cultivars). Fatty acid methyl ester (FAME) profiles of roots and rhizosphere soil of the cultivars were also compared. These crop cultivars represent a continuum from older to the most recent crop cultivars, with Quest being a transgenic canola variety tolerant of the herbicide glyphosate. To the best of our knowledge, Quest is not directly related to Parkland or Excel. The endophytic community of Quest used the BiologTM polymer, carbohydrate, amino acid, and miscellaneous functional guilds at a slower rate than the endophytic community of Excel or Parkland. Furthermore, there were lower levels of the microbial FAMEs, 18:0, 18:3 w6c (6,9,12), 16:0 2OH, and 15:0 2OH in the roots of Quest compared with Excel or Parkland. In contrast, there were no differences in the utilization rate of BiologTM functional guilds or the microbial FAMEs in the roots of the three wheat cultivars studied. The correlation between the ability of endophytic and rhizosphere communities to utilize BiologTM substrates was lower in Quest and CDC Teal compared with earlier crop cultivars. Our results indicate that endophytic and rhizosphere microbial communities of the transgenic cultivar Quest were different from nontransgenic cultivars grown at the same field site.Key words: Brassica spp., Triticum spp., rhizosphere, endophytes, FAME, BiologTM, transgenic.