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Background: Per- and polyfluoroalkyl substances (PFAS) are considered 'contaminants of emerging concern' due to their environmental persistence, bio-accumulative potential, and adverse effects on human health. They are widely employed in producing various goods used in daily life, such as non-stick cookware, cleaning agents, and many industrial applications in aerospace, automotive, construction, electronics, and military, all of which ultimately end up in different environmental matrices. India's rapid economic growth necessitates a comprehensive understanding of its PFAS contamination levels and potential human and ecological exposure. Results: The present study reports the quantification of one long-chain and two short-chain perfluoroalkyl acids and three long-chain and two short-chain perfluorosulfonic acids and qualitative analysis using suspect screening in the waters of Chennai, Tamil Nadu, India. Samples were collected from surface and groundwater sources, including the Adyar river, Buckingham canal, Chembarambakkam lake, and the water treatment plant. The concentrations analyzed in all the samples ranged from 0.10 ng/L to 136.27 ng/L. Groundwater had some of the highest concentrations of PFAS. L-PFBS (up to 136.27 ng/L) and PFOA (up to 77.61 ng/L) are present in all the samples. The concentrations of all the target PFAS increased in the 5 to 103% range in the final treated water compared to the raw water of the water treatment plant. This treated water is distributed as a source of drinking water for the residents of Chennai. Additionally, the dominance of short-chain compounds (4–7 carbons) over long-chain compounds (> 7 carbons) was observed. Suspect screening revealed numerous precursors and other fluorinated compounds abundantly present in the samples. Conclusions: Our study revealed PFAS levels up to 136.27 ng/L in both surface and groundwater samples from Chennai. These findings raise concerns about potential risks to ecosystems and human well-being. The dominance of short-chain PFAS coupled with abundant precursors and unidentified fluorinated compounds indicates an ongoing shift toward alternatives. Conventional water treatment is ineffective in eliminating these chemicals from the water system; rather, increasing the PFAS concentrations from raw water to treated water necessitates advanced polishing steps. Industrial emissions, untreated domestic wastewater discharge, and open dump sites have been suspected as the significant sources of contamination, highlighting the need for further investigation to fully assess the extent of PFAS contamination in Chennai. Highlights: Groundwater and surface waters showed predominant presence of PFOA and L-PFBS. Concentrations of target PFAS increased from raw water to treated water in a conventional WTP. Numerous precursors and other fluorinated compounds are abundantly present in the collected samples. Open dump sites, untreated domestic wastewater discharge, and industrial emissions have been suspected as the contributing sources. [ABSTRACT FROM AUTHOR]