Environmental factors influence the disease susceptibility of crop plants. In this study, we established an experimental system to investigate the effects of vernalisation, temperature and plant growth stage on the susceptibility of winter wheat and winter triticale to Puccinia striiformis, the causal agent of yellow (stripe) rust. Two temperature regimes: standard (18 °C day/12 °C night) and low (12 °C day/6 °C night), vernalised and non-vernalised seedlings, vernalised adult plants and two pathogen races were investigated. At low temperatures, vernalisation reduced the susceptibility of seedlings exposed to the ‘Warrior’ race, while this was only the case for five out of eight varieties exposed to the ‘Kranich’ race. Changing from standard to low temperature resulted in increased susceptibility of non-vernalised seedlings of seven varieties inoculated with the ‘Warrior’ race and five varieties inoculated with the ‘Kranich’ race. Increased susceptibility at low temperature was also detected for several varieties at the adult plant growth stage. Comparisons between vernalised seedlings and adult plants revealed an effect of plant growth stage on disease susceptibility (e.g., Adult Plant Resistance) in five varieties at standard temperature for the ‘Warrior’ race and in five and four varieties at standard and low temperature respectively, for the ‘Kranich’ race. The complex and unpredictable interactions between environment and pathogen influencing yellow rust susceptibility of individual varieties stress the importance of phenotyping for disease resistance under different environmental conditions and pathogen populations. The environmental impact on rust susceptibility should also be taken into account in early-warning systems targeting wheat and triticale breeding programmes and growers.