The classification of disorders such as ambiguous genitalia in newborns is difficult because similar or identical phenotypes could have several different aetiologies. A newborn with ambiguous genitalia needs evaluation to detect life-threatening conditions (salt-losing crisis due to congenital adrenal hyperplasia or Wilms' tumour) and gender assignment. The main factor influencing the sex determination of an embryo is the genetic sex determined by the presence or absence of the Y chromosome. Some individuals carry a Y chromosome but are phenotypically female or have a female karyotype but are phenotypically male. The presence of SRY causes the bipotential gonad to develop into a testis. SRY-positi ve XX males have normal genitalia; in contrast SRY-negative XX males usually have genital ambiguity. A small number of SRY-positive XX males also present with ambiguous genitalia. In this article, we studied the causes and characteristics of ambiguous genitalia in15 children who were referred to a cytogenetic laboratory. Cytogenetic analysis was performed for each case. The cases were analysed by also interphase FISH technique to exhibit exist Y chromosome. Genomic DNA was extracted from peripheral leukocytes collected from a venous blood sample. Molecular analysis of sex-reversed patients led to the discovery of the SRY gene (sex-determining region on Y). We performed molecular genetic analysis for Y chromosomal loci (SRY, ZFY, SY84, SY86, SY127, SY134, SY254, SY255) blood leukocytes. The etiology of ambiguous genitalia is variable. The physician managing these families could minimize the trauma of having a child with unidentified sex by providing appropriate genetic counseling so t hat the parents can make an early decision.