Ambiguous Genitalia (AG) is the birth defect where the gender of an individual is ambiguous. The retrospective study aimed at finding the frequency of sex chromosomal abnormalities and its various patterns in the samples with history of Ambiguous Genitalia. This retrospective study was carried out for two years on 487 cases referred in view of clinical suspicion of AG. The samples were received at the department of cytogenetics, Metropolis Healthcare Ltd, Mumbai, India. Peripheral blood (2-3 ml) from all study subjects was collected in sodium heparin green top vacutainer tube. Both, 48 hours and 72 hours’ cultures were set & analysed by GTG–banding at 450-550 band level. Out of 487 study samples, 243 cases and 220 cases were registered as male and female, respectively. For 24 cases, gender was not mentioned or identified. Out of the total 487 cases, 73cases showed cytogenetic abnormality and normal polymorphic variation was seen in 15 cases. Out of 32 abnormal cases registered as male, mosaic pattern for sex chromosome was seen in 11 cases. Out of 41 abnormal cases registered as females, 4 cases showed mosaic pattern. Since Metropolis Healthcare Ltd is a referral laboratory and because of the selection bias, there could be slight variations in percentage of abnormality as compared to the published data in scientific literature. This study concludes that for all cases of genital ambiguity, a cytogenetic investigation as a basic approach could be very informative and should be suggested.