Ambiguous Genitalia, a rare genetic disorder is caused by defects in the process of fetal sexual determination and differentiation where a newborn needs prompt evaluation to detect lethal conditions and gender assignment. This genetic screening is taken up with an aim to evaluate the prevalence of the patients born with Ambiguous Genitalia existing in our population. We evaluated the patients for Ambiguous Genitalia with Polymerase Chain Reaction (PCR) using SRY gene and cytogenetic analysis for detecting X and Y chromosome was performed by conventional Karyotyping. Of the 50 Ambiguous Genitalia cases, 21 (42.0 %) were detected positive for SRY gene with 46, XY Karyotype and were recognized as males while as rest of cases 29 of 50 (58%) were confirmed as females by negative SRY gene coupled with 46, XX Karyotype. All the 21 cases positive for SRY gene were cytogenetically evaluated with 46, XY except two cases, one of testicular feminization syndrome and another case of AIS where a Karyotype revealed 46, XY but were negative for SRY gene. 09 of the 50 cases (18%) had undetermined sex and among these 05 (55.5%) were found to be negative for SRY gene with 46, XX Karyotype except one case with 47, XX which was an Edward’s syndrome. 04 of the 09 cases (45.5%) were detected as males with positive SRY gene. Overall 04 cases (08.0 %) that were socially recognized as female for two decades were confirmed to be carrying Y chromosome as depicted by positive SRY gene with 46, XY Karyotype. We found that newborn with Ambiguous Genitalia exist in our region in a good number and the cytogenetic method coupled with molecular analysis by PCR are essential to unfold the sex and help in proper assignment of gender in intersex disorders.