Chronic granulomatous disease (CGD) is a rare primary immunodeficiency affecting the innate immune system. Even if functional tests address the diagnosis of CGD, the identification of a molecular defect is essential for counselling family members at risk for being CGD carriers and for prenatal diagnosis. The X-linked form occurs in 65% of CGD patients. It is due to mutations in the CYBB gene, up to 12% of which are caused by large deletions. CGD carriers are usually healthy, and molecular analysis is essential to reveal their carrier status. The aim of this study was to apply a gene dosage approach, using SYBR green quantitative real-time polymerase chain reaction (RT-PCR), to quantify the genomic copy number in carriers and noncarriers of gross deletions covering the region of the CYBB gene. We studied the expression of two different amplification products of the CYBB gene, and the results confirmed a highly reduced expression of the gene in the carrier samples. The results were confirmed by linkage analysis and fluorescence in situ hybridization. Quantitative real-time PCR is fast and simple to perform, and we propose it as a new routine diagnostic approach to detect CGD carriers of deletions covering the region spanning the CYBB gene.