Chronic granulomatous disease (CGD) is a rare inherited disorder of the innate immune system characterized by severe recurrent bacterial and fungal infections. The syndrome is caused by mutation in any of the five genes coding for the components of the NADPH oxidase complex. The most common form of CGD (~69 % of cases) is caused by mutations in the X-linked CYBB gene and is often more severe than the autosomal recessive forms that are caused by mutations in CYBA, NCF-1, NCF-2 or NCF-4. The mutations detected in CYBB, CYBA and NCF-2 are heterogeneous and often family-specific. In contrast, in more than 94 % of patients with NCF-1 deficiency a single mutation, a GT deletion (DGT) of a GT-GT repeat in the start of exon 2, has been identified. This predominance is caused by recombination events involving NCF-1 and highly homologous pseudogenes that co-localize to the same chromosomal region. Here, we give a review of the genetic background of chronic granulomatous disease, describe an algorithm and suitable methods for the molecular diagnosis and discuss the problems one may encounter in the analysis.