The development of glyphosate-resistant canola has provided improved weed-management options for growers, but crop tolerance to glyphosate may be inadequate at later growth stages. In this study, glyphosate-resistant canola 45H28 (RR) was used to determine the effects of glyphosate application timing on yield and yield components at several sites in western Canada. Canola received a single glyphosate applications at the two-leaf, six-leaf, bolting, and early bloom stages and sequential applications at the two-leaf + six-leaf, two-leaf + bolting, and two-leaf + early bloom stages. Contrasts were made between early vs. late, single vs. sequential, and on-label (two to six-leaf stage) vs. off-label (above six-leaf stage). In general, differences between application timings were observed for yield and yield components in 3 of 8 site-yr. Off-label applications of glyphosate (later than six-leaf) significantly decreased yield, seeds per pod, and increased thousand-seed weight and aborted pods in canola at the Lethbridge and St. Albert locations. Increased glyphosate translocation because of adequate, but not excessive, moisture to new growth may have suppressed new seed formation and encouraged pod abortion at the time of application in the 2010 and 2011 seasons. Results from this experiment demonstrate the importance of proper application timing of glyphosate on canola and can help better predict the effects of late applications.