Polyfluoroalkyl substances and perfluoroalkyl substances (PFAS) are a family of anthropogenic chemicals that are used in food packaging, waterproof clothing, and firefighting foams for their water and oil resistant properties. Though levels of some PFAS appear to be decreasing in Canada's south, environmental levels have been increasing in the Arctic due to long-range transport. However, the implications of this on human exposures in sub-Arctic and Arctic populations in Canada have yet to be established. To address this data gap, human biomonitoring research was completed in Old Crow, Yukon, and the Dehcho region, Northwest Territories. Blood samples were collected from adults residing in seven northern First Nations and were analyzed by liquid chromatography mass spectrometry. A total of nine PFAS were quantified: perfluorooctanoic acid (PFOA), perfluorooctane sulphonic acid (PFOS), perfluorohexane sulphonic acid (PFHxS), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), and perfluoroundecanoic acid (PFUdA), perfluorobutanoic acid (PFBA), perfluorohexanoic acid (PFHxA), and perfluorobutane sulphonic acid (PFBS). In the Dehcho (n = 124), five PFAS had a detection rate greater than 50% including PFOS, PFOA, PFHxS, PFNA, and PFDA. In addition to these PFAS, PFUdA was also detected in at least half of the samples collected in Old Crow (n = 54). Generally, male participants had higher concentrations of PFAS compared to female participants, and PFAS concentrations tended to increase with age. For most PFAS, Old Crow and Dehcho levels were similar or lower to those measured in the general Canadian population (as measured through the Canadian Health Measures Survey or CHMS) and other First Nations populations in Canada (as measured through the First Nations Biomonitoring Initiative or FNBI). The key exception to this was for PFNA which, relative to the CHMS (0.51 μg/L), was approximately 1.8 times higher in Old Crow (0.94 μg/L) and 2.8 times higher in Dehcho (1.42 μg/L) than observed in the general Canadian population. This project provides baseline PFAS levels for participating communities, improving understanding of human exposures to PFAS in Canada. Future research should investigate site-specific PFNA exposure sources and monitor temporal trends in these regions.