Quartet pollen, where pollen grains remain attached to each other post-meiosis, is useful for tetrad analysis, crossover assessment and centromere mapping. We observed the quartet pollen phenotype for the first time in the agriculturally significant Brassica genus, in an experimental population of allohexaploid Brassica hybrids derived from the cross (Brassica napus × B. carinata) × B. juncea followed by two self-pollination generations. Quartet pollen production was assessed in 144 genotypes under glasshouse conditions, following which a set of 16 genotypes were selected to further investigate the effect of environment (warm: 25 °C and cold: 10 °C temperatures) on quartet pollen production in growth cabinets. Under glasshouse phenotyping conditions, only 92 out of 144 genotypes produced enough pollen to score: of these, 30 did not produce any observable quartet pollen, while 62 genotypes produced quartet pollen at varying frequencies. Quartet pollen production appeared quantitative and did not clearly fall into phenotypic or qualitative categories indicative of major gene expression. No consistent effect of temperature on quartet pollen production was identified, with some genotypes producing more and some producing less quartet pollen under different temperature treatments. The genetic heterogeneity and frequent pollen infertility of this population prevents strong conclusions being made. However, it is clear that the quartet phenotype in this Brassica population does not show complete penetrance and shows variable (likely genotype-specific) response to temperature stress. In future, identification of quartet phenotypes in Brassica would perhaps best be carried out via screening of diploid (e.g. B. rapa) TILLING populations.