Polyunsaturated fatty acids (PUFAs), including the long-chain (LC) ω-3 PUFAs, are important for health. The aim was to assess if the Anti-Cancer Council of Victoria Dietary Questionnaire (ACC DQ) accurately determines PUFA intakes compared with the recently validated electronic PUFA food-frequency questionnaire (FFQ). Forty-one study volunteers were recruited from the local Illawarra region of New South Wales, Australia. The method of triads was used to determine validity coefficients by comparing the ACC DQ intakes against a 3-d weighed food record and appropriate blood biomarkers (erythrocytes and plasma fatty acids). These validity coefficients were subsequently compared with previously published validity coefficients from the PUFA FFQ. Using erythrocytes as the biomarker, the electronic PUFA FFQ had much higher validity coefficients compared with the ACC DQ for eicosapentaenoic acid (0.92 versus 0.19), docosahexaenoic acid (0.69 versus 0.26), and total LC ω-3 PUFAs (0.78 versus 0.23), respectively, whereas ω-6 PUFAs were comparable. Using plasma as the biomarker, the electronic PUFA FFQ had much higher validity coefficients compared with the ACC DQ for α-linolenic acid (0.96 versus 0.49), eicosapentaenoic acid (0.87 versus 0.19), docosahexaenoic acid (0.64 versus 0.24), and total LC ω-3 PUFAs (0.73 versus 0.21), respectively, whereas ω-6 PUFAs were comparable. The validated electronic PUFA FFQ is better suited to determine ω-3 PUFA intakes than the ACC DQ.