The pathogenic fungal species Pyrenophora tritici-repentis (Ptr) and Parastagonospora nodorum (Pan) are common in many wheat-producing parts of the world. These two fungi cause tan spot and septoria nodorum blotch, respectively, frequently co-infecting wheat leaves. Empirical studies of this and other co-infections are rare because of the visual similarity of symptoms and the lack of robust methods for quantifying the abundance of pathogens associated with the co-infection. Here, we use a recently developed molecular method that simultaneously distinguishes and quantifies, in DNA equivalent, the abundance of Ptr and Pan, thereby allowing the prevalence of co-infection to be determined. The study examines the prevalence of co-infection under field conditions, at three widely spaced sites and on three wheat (Triticum aestivum L.) cultivars varying in disease resistance. Co-infection by Ptr and Pan was almost ubiquitous (overall prevalence 94%), and Pan DNA was detected only in association with Ptr. Although Ptr and Pan commonly co-infected, Ptr was more abundant during early and mid-season, at 80% of total fungal abundance when crops were tillering and 67% at booting stage. Pan became as abundant as Ptr when crops reached flowering. Variability in total fungal abundance and disease severity was primarily determined by cultivar; however, Ptr was the more abundant despite differences in cultivar resistance to this pathogen.