|There is a growing interest in determining fatty acid reference intervals from pregnancy cohort, especially considering the lack of reference values for pregnant women in the literature and the generalized misconception of equating reference intervals for nonpregnant women as equivalent to pregnant women. Seafood and supplements are important dietary sources for the omega-3 long-chain polyunsaturated fatty acids (ω-3 LCPUFA), such as eicosapentaenoic acid (EPA, 20:5ω-3), docosapentaenoic acid (DPA, 22:55ω-3), and docosahexaenoic acid (DHA, 22:6ω-3). Sufficient intake of EPA and DHA is vital during pregnancy for the development of the fetus, as well as for maintaining adequate levels for the mother. This study describes the fatty acid status and suggests reference values and cut-offs for fatty acids in red blood cells (RBC) from pregnant women (n = 247). An electronic food frequency questionnaire (e-FFQ) mapped the dietary habits of the participants, and gas chromatography was used to determine the fatty acid levels in RBC. The association between e-FFQ variables and fatty acid concentrations was established using a principal component analysis (PCA). Twenty-nine-point-one percent (29.1%) of the participants reported eating seafood as dinner according to the Norwegian recommendations, and they added in their diet as well a high percentage (76.9%) intake of ω-3 supplements. The concentration levels of fatty acids in RBC were in agreement with those reported in similar populations from different countries. The reference interval 2.5/97.5 percentiles for EPA, DPA, DHA were 0.23/2.12, 0.56/2.80, 3.76/10.12 in relative concentration units (%), and 5.99/51.25, 11.08/61.97, 64.25/218.08 in absolute concentration units (µg/g), respectively. The number of participants and their selection from all over Norway vouch for the representativeness of the study and the validity of the proposed reference values, and therefore, the study may be a useful tool when studying associations between fatty acid status and health outcome in future studies. To the best of our knowledge, this is the first PCA study reporting a direct association between ω-3 LCPUFA and intake of seafood and ω-3 supplements in a pregnancy cohort.