A real-time quantitative PCR technique has been used to develop a rapid and sensitive seed health test for Pyrenophora spp. on barley seed. Using the fluorescent reporter dye SYBR Green I for real-time detection of PCR amplification, pathogen DNA extracted from infected seed can be quantified to the picogram level. The amount of Pyrenophora DNA extracted from seed samples of an artificial infection level gradient, constructed by mixing infected and uninfected seed, correlated with good agreement (r = 0.931) to percentage infection levels of the same samples measured by agar plate testing. In addition, a correlation of r = 0.883 was obtained between the two testing methods for naturally infected seed, ranging from 0% to 89% infection. Samples could be quantified to below the 2% voluntary threshold required for deciding on seed treatment. The proposed test was performed in three parts: (i) quantification of Pyrenophora spp. infection using Pyrenophora-specific PCR primers; (ii) test of any negative samples from (i) with barley-specific PCR primers to check the DNA extraction process; (iii) test of positive samples from (i) for the presence of Pyrenophora graminea using P. graminea-specific PCR primers. All PCRs were performed in the LightCycler™ instrument allowing each PCR run and analysis to be completed within 30 min. With the current daily receipt of samples (batches up to 16) the test can be completed in 8 h, compared to 7 days for the traditional agar plate test.