Abstract: Objective　To explore the genetic characteristics of 21 hydroxylase deficiency (21-OHD) in Henan area. Methods　The genetic test results of 21-OHD children and their families of Han nationality originally from Henan province were retrospectively analyzed from 2009 to 2016. Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) methods were performed to detect mutations in CYP21A2 gene. For children with negative mutation of CYP21A2 gene, next generation sequencing (NGS) was performed to confirm the diagnosis. Results　A total of 55 patients from 54 families with a disease course of one month to 8 years were studied. There were 49 cases (37 boys and 12 girls) of clinically diagnosed salt wasting (SW) type and 6 cases (3 boys and 3 girls) of simple virilizing (SV) type. The CYP21A2 gene mutation was detected in 46 children, and the most frequent mutation identified was c.293-13A/C>G (I2G), followed by partial or complete deletion of the gene, c.955G>T (p.Q319X), c.515T>A (p.I172N) and c.1069G>T (p.R357W). Among 9 children with negative CYP21A2, there were 7 children whose diagnosis was corrected to congenital adrenal dysplasia due to positive NROB1 (DAX1) mutations detected by NGS analysis. Conclusions　The most common mutation type of CYP21A2 in children diagnosed with 21-OHD clinically in Henan is c. 293-13a/c >G (I2G), followed by the large fragment deletion of the gene.