Heat stress can frequently limit the yield of Brassica napus L. grown in Canada because of the often unavoidable concurrence of high temperatures and flowering. Ten B. napus inbred genotypes, an open-pollinated B. napus commercial cultivar and a B. juncea genotype were grown in a greenhouse and subjected to two temperature regimes in a growth chamber for 14 days during flowering: control 22°C/10°C and high 31°C/14°C (day/night). Floral buds were sampled at the end of the 14-day treatments, and an untargeted metabolomic assessment was completed using gas chromatography–mass spectrometry. Flower duration, number of flowers, number of pods, biomass, number of seeds and seed weight were recorded. Yield was reduced by 55% in the heat treatment during winter and by 41% during the subsequent autumn experimental run. Of the 12 genotypes, five were classified as heat-tolerant and four as heat-susceptible based on the calculated heat susceptibility index across two experiments. In total, 25 metabolic markers were identified that discriminated between the heat-tolerant and -susceptible genotypes exposed to the heat treatment. The variation identified within this set of germplasm has provided evidence that variation exists within B. napus to enable genetic gain for heat tolerance.