Septoria leaf spot is one of the most widespread diseases affecting pistachio (Pistacia vera) in countries of the Mediterranean region.Septoria pistaciarumwas recently confirmed as the causal agent of this disease in Italy. Currently, the detection ofS.pistaciarumrelies on isolation techniques. These require significant amounts of labor, and time for completion. Also, a reliable identification requires the sequencing of at least two housekeeping genes, in addition to the morphological observations. To accurately detect the presence and quantifyS.pistaciarumin pistachio tissues, a molecular tool was necessary. We designed applicable primers that allow reliable amplification of the β-tubulin gene. The amplification of target DNA was highly efficient, with a 100% success rate, and the assay was able to detect as little as 100 fg/rxn of pure fungal DNA. When tested in artificial mixtures of plant and pathogen DNAs, the assay was able to detect the pathogen consistently at a limit of detection of 1 pg/rxn. The assay was also effective in identifying the pathogen in naturally infected samples, providing rapid detection in all symptomatic specimens. The resulting qPCR assay is an improved detection tool for accurate diagnosis ofS.pistaciarumthat can also contribute to better understand the population dynamics of the pathogen in the orchard.