Mutations in NCF1(p47phox) cause autosomal recessive chronic granulomatous disease (CGD) with abnormal dihydrorhodamine (DHR) assay and absent p47phoxprotein. Genetic identification of NCF1mutations is complicated by adjacent highly conserved (>98%) pseudogenes (NCF1Band NCF1C). NCF1has GTGT at the start of exon 2, whereas the pseudogenes each delete 1 GT (ΔGT). In p47phoxCGD, the most common mutation is ΔGT in NCF1(c.75_76delGT; p.Tyr26fsX26). Sequence homology between NCF1and its pseudogenes precludes reliable use of standard Sanger sequencing for NCF1mutations and for confirming carrier status. We first established by flow cytometry that neutrophils from p47phoxCGD patients had negligible p47phoxexpression, whereas those from p47phoxCGD carriers had ∼60% of normal p47phoxexpression, independent of the specific mutation in NCF1. We developed a droplet digital polymerase chain reaction (ddPCR) with 2 distinct probes, recognizing either the wild-type GTGT sequence or the ΔGT sequence. A second ddPCR established copy number by comparison with the single-copy telomerase reverse transcriptase gene, TERT. We showed that 84% of p47phoxCGD patients were homozygous for ΔGT NCF1. The ddPCR assay also enabled determination of carrier status of relatives. Furthermore, only 79.2% of normal volunteers had 2 copies of GTGT per 6 total (NCF1/NCF1B/NCF1C) copies, designated 2/6; 14.7% had 3/6, and 1.6% had 4/6 GTGT copies. In summary, flow cytometry for p47phoxexpression quickly identifies patients and carriers of p47phoxCGD, and genomic ddPCR identifies patients and carriers of ΔGT NCF1, the most common mutation in p47phoxCGD.