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Background: Seafood (fish and shellfish) is an excellent source of several essential nutrients for pregnant and lactating women. A short food frequency questionnaire (FFQ) that can be used to quantitatively estimate seafood consumption would be a valuable tool to assess seafood consumption in this group. Currently there is no such validated FFQ in Norway. Objective: The objective of this study was to establish and validate a seafood index from a seafood FFQ against blood biomarkers (the omega-3 index, the omega-3 HUFA score, and serum 25OH vitamin D). Design: We assessed maternal seafood consumption during the 28th gestation week in healthy Norwegian women (n=54) with a seafood FFQ. A seafood index was developed to convert ordinal frequency data from the FFQ into numerical scale data. The following blood biomarkers were used as a validation method: omega-3 index, omega-3 HUFA score, and the serum 25OH vitamin D. Results: The reported frequency of seafood as dinner and as spread was strongly correlated with the estimated frequencies of seafood as dinner and as spread. This indicated that the seafood index is a valuable tool to aggregate reported frequencies from the seafood FFQ. The seafood index composed of the frequency of seafood consumption and intake of omega-3 supplements, termed the total seafood index, correlated positively with the omega-3 index, omega-3 HUFA score, and 25OH vitamin D. Conclusion: We established and validated a seafood index from a seafood FFQ. The developed seafood index can be used when studying health effects of seafood consumption in large populations. This seafood FFQ captures seafood consumption and omega-3 supplement intake considerably well in a group of pregnant women.