Purpose: Normal penile development depends on testosterone biosynthesis, its conversion via steroid 5α-reductase type 2 to dihydrotestosterone, and functional androgen receptor. Mutations in the 5α-reductase type 2 (SRD5A2), androgen receptor (AR), and 3-beta-hydroxysteroid dehydrogenase type 2 (HSD3B2) gene impair masculinization and may be associated with ambiguous genitalia in 46,XY disorders of sex development (DSD). This genetic study was conducted to test these hypotheses and aid in accurate diagnosis. Methods: We analyzed the SRD5A2, AR, and HSD3B2 genes in 98 Korean patients with 46,XY DSD. The median age was 2 years (range, 1 month-18 years). The control group included 80 males. We applied polymerase chain reaction and direct sequencing to analyze the coding regions of SRD5A2, AR, and HSD3B2 genes. The effects of the identified mutations were assessed using in silico modeling tools, such as PolyPhen-2, SIFT, and PROVEAN. Results: Based on various in silico tools, 4 SRD5A2 missense single nucleotide polymorphisms (SNPs), and 2 AR missense SNPs may affect protein functions. Among these six SNPs, SRD5A2:rs121434244 (R246W), SRD5A2:rs9332964 (R227Q), and AR:rs137852569 (A597T) were previously reported pathogenic mutations. The other three mutations, SRD5A2:rs9332961 (G203S), SRD5A2:rs9332967 (R246Q), and AR:rs200185441 (Q58L), which showed possible association with 46,XY DSD, had not been reported in literature to date. Conclusion: Our data demonstrated that several mutations in SRD5A2 and AR genes were associated with ambiguous genitalia. Genetic studies may play an important role for accurate diagnosis and further treatment of micropenis, hypospadia, or other ambiguous genitalia.