In recent years, environmental issues emerging from per- and polyfluoroalkyl substances (PFAS) have raised high concern worldwide. Levels of human exposure to PFAS remain unknown in India. Biomonitoring data obtained from hair analysis have been evidenced to provide insight into retrospective human exposure to PFAS. In this study, 25 PFAS, including perfluoroalkyl acids and their precursors, were measured in 39 human hair samples collected from 14 cities in India. The inuﬂence of gender on the PFAS levels was also examined. To our knowledge, this is the first attempt to provide preliminary indicative data (due to the limited sample size and variability in hair-length sampling) on the levels of PFAS in Indian hair. The concentrations of total PFAS in hair varied from below matrix-specific limit of quantiﬁcation (<0.02 ng/g) to 3.78 ng/g. Among 9 PFAS quantified, perfluorohexanesulfonic acid (PFHxS), perfluorooctanesulfonic acid (PFOS), and perfluorooctanoic acid (PFOA) were the predominant compounds. Categorized into 4 regions, PFAS contamination exhibited certain regional difference where South India may show higher levels than the other regions. Highly significant positive correlation was observed between PFHxS and PFOS (p ≪ 0.001; r = 0.644), suggesting similar pathways of exposure to the two compounds. Higher PFAS occurrence was generally observed in the hair of females. Our results highlighted the urgent need to investigate the deposition mechanism of PFAS in hair.