A higher proportion of n-3 long-chain PUFA in tissue lipids has been associated with a lower risk of CVD and some cancers. Diet is an important predictor of n-3 long-chain PUFA composition; however, the importance of non-dietary factors such as sex and age is unclear. We measured the proportion of n-3 long-chain PUFA in serum phospholipid, cholesterol ester and TAG of 2793 New Zealanders 15 years or older who participated in the 1997 National Nutrition Survey to determine differences by sex and age. Women had lower proportions of EPA and docosapentaenoic acid in phospholipid, by 0.07 (P = 0.004) and 0.10 (P < 0.001) mol%, respectively, and a higher proportion of DHA by 0.16 mol% (P = 0.001) compared with men. Intake of fish fat did not differ between men and women. There was a positive association between age and the proportion of EPA and DHA in phospholipid (P < 0.001). The sex differences in EPA and DHA were similar at all ages. Similar sex and age differences in serum cholesterol ester n-3 long-chain PUFA were found; only age differences were found in serum TAG. Sex and age differences in n-3 long-chain PUFA occur in the general population. Men and women may need to be considered separately when examining the association between disease risk and biomarkers of n-3 fatty acids.