Ambiguous genitalia, a disorder of sex development (DSD), are a group of conditions where normal development of the reproductive organs and genitals are not seen. The term DSD was given to congenital conditions that are atypical showing differences in development of chromosomal, gonadal,or anatomical sex. Ambiguous genitalia are a mixture of male and female sexual characters found in a person. In the present studywe have considered the gene, Nuclear receptor subfamily 5, group A, member 1 (NR5A1). This gene is responsible for the production of transcription factor, steroidogenic factor-1 (SF-1), which plays an important role in the development of gonads and the adrenal glands. A mutation on this gene is associated with ambiguous genitalia. The objective of this study is to find the mutational analyses in ambiguous genitalia patients. The DNA was extracted from blood samples of 12 patients and analysed using bio-photometer. PCR technique was used to amplify the NR5A1 region and confirmed using SSCP. Further confirmations will be done by sequencing analysis. © 2014, Sphinx Knowledge House. All rights reserved.