Ambiguous genitalia is seen as the most common phenotypic reflection of sexual development disorders. Congenital adrenal hyperplasia (CAH) is the most common cause of ambiguous genitalia, while the most common cause of CAH is a 21-hydroxylase deficiency with a rate of 90-95%. The disease is caused by mutations in the CYP21A2 gene located at 6p21.3. It is inherited in an autosomal recessive manner. Seven previously identified point mutations, an 8-bp deletion and large deletions, have significant role in the etiology of the disease. In this study, we aimed to report CYP21 molecular genetic evaluation by RFLP and MLPA methods in classic CAH patients with 21-hydroxylase deficiency. In this study, 26 patients with pre-diagnosis of Classic Type Congenital Adrenal Hyperplasia due to 21-Hydroxylase deficiency were reported. Seven previously identified point mutations, an 8-bp deletion, and large deletions were analyzed by PCR-RFLP methods in the patient group. For the MLPA study, SALSA MLPA KIT P050-B2 CAH (Lot0408) kit which was produced by MRC Holland was used. In 21 (80.7%) of 26 patients analyzed, causative mutations were found. The most frequent mutation was the large deletions (6 patients, 12 allels), accounting 23% of the patients. In 21 (80.7%) of 26 patients, the causative mutations were found by using PCR (8-bp del. and large deletions) and RFLP (7 known point mutations) methods. MLPA analysis confirmed all of the deletions detected by PCR-RFLP, and the 83% of the detectable point mutations with MLPA. A complete genotype-phenotype relationship could be established in all patients in whom mutation could be detected in the study group.