Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders mainly caused by a defect in the steroid 21-hydroxylase gene (CYP21A2). In this study, we investigated the molecular defects of 25 Chinese pedigrees with 21-hydroxylase deficiency (21-OHD). Diagnosis of the probands in the families was based on their typical clinical presentations, such as inborn ambiguous genitalia, or early onset of salt wasting and biochemical metabolite abnormalities. All 10 exons and exon-intron boundaries of the CYP21A2 gene were amplified from the genomic DNA of the probands and then analyzed by direct sequencing. The phenotypes of the 26 patients from 25 pedigrees were classified as the classical form of 21-OHD. One novel mutation (c.1223 G>T) and 13 recurrent mutations of CYP21A2 were identified in the 25 pedigrees by genetic analysis. The novel c.1223 G>T mutation results in the substitution of arginine by leucine at amino acid position 408 (p.Arg408Leu). The most frequent mutation alleles were IVS2-13A/C>G (14/52) and I172N (11/52), followed by chimeric mutations (10/52). Forty six of 52 mutated alleles resulted from pseudogene conversion and 6 of 52 from random mutations. The spectrum of CYP21A2 mutation in our study was slightly different from those previously reported in Chinese and in other ethnic groups of the world. Although microconversion events were the main cause of mutations in the CYP21 gene, random mutations with a common origin can also be the reason for 21-OHD.