The aim of our study was to perform a comprehensive CYP21A2 mutation analysis in 113 21-OHD patients and their parents using CNVplex, SNaPshot assay and Sanger sequencing based on locus-specific PCR in a sequential strategy to provide fast, reliable and comprehensive genetic diagnoses. The gene discussed is CYP21A2; the disease is classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency.