If predictions are correct, heat stress during reproduction will become a yield limiting factor in many world crops and breeding heat stress tolerance a major goal. The objective of our paper was to highlight a novel system to investigate the influence of temperature (T) on pollen germination using a thermal gradient PCR programmed to establish differential Ts across 12 wells of a PCR plate. Seven cultivars of Brassica napus L. were grown through flowering in a cool growth cabinet (20/15°C day/night) or a heat stress cabinet (HST, 27/22°C day/night). Pollen from each cultivar × cabinet combination was aspirated from 6 opened flowers, and suspended in germination media. Drops of the pollen suspension were floated on media in each well, and the PCR T was set to 30°C with a gradient of ± 10°C, creating a range from ∼20 to 40°C from left to right. After an 8 h treatment, the pollen germination (pg, %) and pollen tube growth score (ptg, 1–5) were evaluated using a microscope. There were significant differences among cultivars for pg and ptg score and significant differences among well T for pg and ptg score. Pollen tubes grew best at T from 20 to 23°C. Well T exceeding 33°C reduced pg and ptg score, although 3 of the 8 cultivars had good pg even at 36°C. HST >29°C, in a growth cabinet, generally resulted in B. napus raceme sterility, although our experiment showed that pollen was still capable of germinating up to 33°C, indicating that pollen germination may not be the only reason for heat stress susceptibility.