In order to detect and quantify Septoria tritici infection levels in wheat leaves, a polymerase chain reaction (PCR) assay was developed using the β-tubulin gene as target. Specific PCR primers were designed by aligning and comparing β-tubulin sequences from other fungi. The final primer set was selected after being tested against several fungi, and against S. tritici-infected and uninfected wheat leaves from different localities. A single DNA fragment (496 bp) was amplified from S. tritici, whereas no products were generated from DNA of the host plant or other micro-organisms associated with wheat leaves. Using agarose gel analysis, approximately 2 pg S. tritici genomic DNA could be detected in each assay. However, for rapid quantification of PCR-amplified products, a fluorometric microtitre plate-formatted PicoGreen assay was used; this could detect as little as 10 pg S. tritici DNA in the presence of 200 ng wheat leaf DNA. The PCR/PicoGreen assay was applied successfully to study the colonization, infection and subsequent disease development of S. tritici on wheat, both under controlled conditions in the glasshouse and in the field.