Congenital adrenal hyperplasia (CAH) is a common autosomal recessive metabolic disease, mainly resulting from a mutation in the steroid 21 hydroxylase (CYP21) gene. CYP21 gene is located in the HLA class III region on the short arm of the chromosome 6 (6p21.3) in tandem with a highly homologous pseudogene (CYP21P). Several techniques for detection of CYP21 point mutations have been used; however, contamination by the highly homologous pseudogene sequence is a major problem. Aim of this study was to introduce a method consisting of differential polymerase chain reaction (PCR) amplification of the CYP21 gene and non-functional CYP21P gene using specific primers. A second PCR was then performed using a panel of specific primers followed by restriction enzyme analysis for each of the examined mutations associated with CAH. Total of 34 individuals (patients and family members) were analyzed. Splicing mutation I2 was detected in 11 individuals (8 homozygotes and 3 heterozygotes), mutation in exon 8 (codon 318) in 8 (2 homozygotes and 6 heterozygotes), mutation in exon 1 (codon 30) in 7 (2 homozygotes, 5 heterozygotes), mutation in exon 7 (codon 281) in 5 individuals (heterozygotes), and mutation in exon 8 (codon 356) in 1 (heterozygote). Most common was splicing I2 mutation (27, 94%) followed by exon 8 mutation (codon 318) - 21.74% of analyzed chromosomes and exon 1 (codon 30) was detected in 15% of explored chromosomes. This approach provides detection of specific CYP21 mutation, as well as detection of zygosity. This method is easy to perform, results are obtained in a short time, and it is appropriate as a diagnostic tool in a clinical practice and for prenatal diagnosis.