Fungal diseases of wheat are among the most damaging plant pathogens, causing significant losses of total grain yield if left untreated. Parastagonospora nodorum (Pn) and Pyrenophora tritici-repentis (Ptr) cause septoria nodorum blotch (SNB) and tan spot (TS) of wheat, respectively. Pn and Ptr can co-exist in the same lesion of infected wheat and are non trivial to distinguish from one another based on physical disease symptoms. Digital droplet PCR (ddPCR) is a high throughput and highly sensitive polymerase-based assay that allows for the quantification of SNP-level specific DNA sequences. We have developed a fluorescent probe-based assay capable of single well, reference-free simultaneous quantification of SNB and TS during host infection. The assay shows no off-target effects across a wide range of closely related cereal necrotrophs and can resolve pathogen titre at up to 5pg/µl of purified fungal gDNA, or 1ng/µl of infected plant tissue. Based on a hypervariable region within the highly conserved α-tubulin gene, the assay can be easily adapted for studying other cryptic plant pathosystems, and will provide another tool for dissecting the relationship between pathogens as well as with their hosts. We have also exploited ddPCR to examine competition between pathogen isolates with differences in effector gene expression profile of the same species during infection on wheat. The implications of this study will be discussed.