BACKGROUND: Australia's long running human biomonitoring (HBM) program has provided information on per and poly-fluoroalkyl substances (PFAS) serum concentrations in the general population since 2002. The program is based on pooling and analysis of surplus, de-identified, pathology specimens predominantly sourced from Australia's north-eastern state Queensland (QLD). To date, potential nationwide spatial differences across Australia have not been assessed. AIM: The aim of this study was to assess spatial variation of background PFAS serum concentrations across all Australian States and Territories, and to assess if the long running HBM program, representing samples biased towards QLD, can be considered a national reference. METHODS: Surplus pathology serum samples were collected and stratified by States/Territories based on postcode. Pools representing three age groups (5-15, 31-45 and ≥ 60 years), stratified by sex were created. Up to two pools for each age/sex strata, consisting of up to 100 individuals, were created for Australian States and major Territories. Samples were analysed for PFAS using high-performance liquid chromatography-mass-spectrometry. RESULTS AND DISCUSSION: There was a high degree of consistency in the PFAS serum concentration for a given age/sex among pools from the different States/Territories, particularly for perfluoro carboxylic acids. This suggests that PFAS serum concentrations and associated exposure is relatively consistent across Australia. PFAS concentrations measured in QLD pools were not statistically different from the national average, suggesting that the current Australian HBM program can be considered as a national reference of background PFAS serum concentrations.