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Aim: To validate the polyunsaturated food frequency questionnaire (PUFA FFQ) and test for reproducibility in people with end stage renal disease on dialysis treatment. Methods: Participants (n = 32) completed the PUFA FFQ and three 24‐hour recalls. Erythrocyte samples (n = 29) were used for erythrocyte fatty acid analysis. The triangular relationship between the PUFA FFQ, 24‐hour recalls and the biomarker was assessed using the method of triads. Agreement between the two dietary methods was also assessed using Bland–Altman plots and classification by quintiles. Reproducibility was tested on a subset of the group (n = 8). Results: The PUFA FFQ was a valid measure of all PUFA except for docosapentaenoic acid (DPA) and arachidonic acid (AA). Strong validity coefficients were found for n‐3 long‐chain PUFA (LCPUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) of 0.914 (95% CI: 0.665, 0.997) and 0.889 (95% CI: 0.706, 0.994), respectively. In the Bland–Altman plots 91–100% of observations fell between the limits of agreement for all PUFA. There were significant correlations between the initial FFQ and the repeat FFQ for all PUFA except DPA and AA. Conclusions: The PUFA FFQ is a valid tool for assessing PUFA intake in people with end stage renal disease. [ABSTRACT FROM AUTHOR]