Unconventional T cells exist as minor subsets of T cells in peripheral blood. Despite their low number they play a crucial role in various immune-mediated diseases such as inflammation, autoimmunity, allergy, and cancer. Gender-based frequency of these cells altogether on the large number of healthy individuals are unestablished creating hurdles to manifest association with various immune-mediated pathological conditions. In this study, we used a multicolor flow cytometric panel to identify iNKT cells, γδ T cells, and MAIT cells altogether in a total of 94 healthy adult males (44 ± 14 years) and 109 healthy adult females (45 ± 14 years), peripheral blood samples of the Caucasian population. The results revealed iNKT cells in females were 0.273 ± 0.506, higher in number than in males 0.189 ± 0.518 and found to be negatively correlated with age in females. Whereas γδ T cell frequency in males was 3.513 ± 3.048, higher in number than females 2.783 ± 2.657 and each gender was negatively correlated with age. MAIT cells didn’t show any statistically significant difference between genders and were found to be negatively correlated with age. The results of our finding could be used for further gender-wise investigation of various pathological conditions such as cancer and their prognosis, autoimmune diseases, allergies and their pathogenicity.