Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most important diseases of wheat worldwide. Understanding the survival of Pst during the overwintering period is critical for predicting Pst epidemics in the spring. Real-time quantitative PCR (qPCR) methods quantifying Pst DNA and RNA (cDNA) were developed and compared for the ability to quantify viable Pst in leaf tissues. Both qPCR of DNA and RNA can provide reliable measurement of viable Pst in plant tissues prior to the late sporulation stage for which qPCR of DNA gave a much higher estimate of fungal biomass than qPCR of RNA. The percentage of Pst biomass that was viable in detached and attached leaves under low temperatures decreased over time. Pst survived longer on attached leaves than on detached leaves. The survival of Pst in cultivars with strong winter-hardiness at 0°C and -5°C was greater than those with weak winter-hardiness. However, such differences in Pst survival among cultivars were negligible at -10, -15 and -20°C. Results indicated that Pst mycelia inside green leaves can also be killed by low temperatures rather than through death of green leaves under low temperatures. The relationship of Pst survival in attached leaves with temperature and winter-hardiness was well described by logistic models. Further field evaluation is necessary to assess whether inclusion of other factors such as moisture and snow cover could improve the model performance in predicting Pst overwintering potential, and hence the epidemic in spring.