Tan spot disease is caused by Pyrenophora tritici-repentis (Ptr), one of the major necrotrophic fungal pathogens that affects wheat crops globally. Extensive research has shown that the necrotrophic fungal effectors ToxA, ToxB and ToxC underlie the genetic interactions of Ptr race classification. ToxA and ToxB are both small proteins secreted during infection, however the structure of ToxC remains unknown. In line with the recent discovery of ToxC1 gene that is involved in ToxC production, a subset of 68 isolates collected from the Australian wheat cropping regions were assessed for presence of all three effectors by pathotyping against four tan spot wheat differential lines and PCR amplification of ToxA, ToxB and ToxC1. Based on the disease phenotypes, the 68 isolates were grouped into two races with 63 classified as race 1 and five as race 2. A representative selection of each race was tested against eight Australian commercial wheat cultivars and showed no distinction between the virulence levels. Sequencing of ToxA showed that both races had identical gene sequences of haplotype PtrA1. All the race 1 isolates possessed ToxC1 but three race 2 isolates also contained ToxC1 despite being unable to induce a spreading chlorotic symptom on the ToxC differential line. QTL mapping confirmed the absence of the ToxC-Tsc1 association in disease response caused by the ToxC1-containing race 2 isolate, however ToxC1 expression was detected during plant infection. Altogether, these results suggest that there is a complex regulatory process involved in the production of ToxC within the Australian race 2 isolates.