Omeag-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) play a critical role in supporting diverse physiological and psychological health outcomes(1). Given their importance, accurate assessment of dietary intake of n-3 LCPUFA is essential for research and policy applications. A PUFA food frequency questionnaire (PUFA FFQ) was previously developed and validated to estimate fatty acid intake for healthy adults in Australia(2, 3). However, recent changes in the availability and diversity of foods and supplements in Australia necessitated an adaptation and re-validation of the tool. The aim of this study was to update and re-validate the PUFA FFQ, assessing its validity, reproducibility and potential for systematic bias. Healthy participants (n = 50) from the Illawarra region in NSW, Australia, provided fasted whole blood samples using a finger stick method (contact-activated lancet) for the measurement of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and total n-3 LCPUFA. Samples were analysed at the Fatty Acid Labs (Victoria, Australia). Power calculations determined that 50 subjects were sufficient to detect an effect size of 1 with > 99% power at a significance of 0·05, assuming a correlation of 0.6 between methods(2). Participants also completed the updated PUFA FFQ and three non-consecutive 24-hour dietary recalls through Intake24, a web-based dietary assessment platform. To assess reproducibility, a second PUFA FFQ (FFQ2) was completed three months after the baseline clinic visit (n = 34), using Wilcoxon Signed Rank tests and Spearman’s correlation coefficients. Relative validity was assessed using the method of triads, which utilises the triangular relationship between the PUFA FFQ, dietary recalls and biomarker data (whole blood EPA, DHA and total n-3 LCPUFA) to estimate the validity coefficients with ‘true intake’. Additional Bland-Altman plots were used to assess absolute agreement. Methods of triads analysis showed effective validity coefficients; for EPA (r = 0.67, 95% CI: [0.27‚ 1.00]), DHA (r = 0.52, 95% CI: [0.26, 0.85]) and total n-3 LCPUFA (r = 0.61, 95% CI: [0.3‚ 1.00]). Bland-Altman plots depicted no systematic error and good agreement between PUFA FFQ and 24-hour dietary recalls, with ³ 97.3% of intakes falling within the limits of agreement (mean difference ± 2 SD). Reproducibility of PUFA intake between FFQ1 and FFQ2 demonstrated no significant differences, with EPA (W = 157.5, p = 0.563), DHA (W = 187, p = 0.297), or total n-3 LCPUFA (W = 223.5, p = 0.649). Spearman’s correlation coefficients for EPA (ρ = 0.921, p < 0.001), DHA (ρ = 0.940, p < 0.001), and total n-3 LCPUFA (ρ = 0.932, p < 0.001), suggesting high reproducibility between the two FFQs. In conclusion, the updated PUFA questionnaire is valid, has no systematic bias and is reproducible, confirming its suitability for assessing n-3 LCPUFA intakes in healthy Australian adults.