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Background: Estimates for fish and shellfish intake are used to inform communities and healthcare systems about potential health risks and benefits for individuals, communities, and vulnerable populations. A dietary assessment instrument was designed for use in populations of high-end consumers of seafood to examine intake of finfish, shrimp, oysters, and blue crab in coastal communities across the Gulf of Mexico., Objective: To validate the reliability of a novel food frequency questionnaire (FFQ) for seafood intake., Design: Test-retest reliability of the FFQ, which included a species-specific photographic portion guide, was evaluated by the inperson administration and readministration of the instrument with each participant by the same interviewer. Responses from coastal and noncoastal participants were compared to discern FFQ reliability in heterogeneous samples. Participants/setting . A convenience sample of 27 coastal participants from Cedar Key, Steinhatchee, and Apalachicola, Florida, reported data for 101 household members; and 15 noncoastal participants from Gainesville, Florida, reported for 42 household members. Analysis . Repeated measures from the FFQ were evaluated using correlation concordance for continuous variables (age, weight, and height) and kappa coefficient for categorical variables (type, amount, and frequency of seafood consumed)., Results: Concordance correlation coefficient (1.00) and kappa coefficient ( r  = 0.73 to 1.00) for yearly and seasonal seafood consumption indicated substantial to almost perfect reproducibility, i.e., participants provided responses that were reproducible. Test-retest agreement was highest for coastal participants who consumed more seafood, as compared to occasional, noncoastal consumers, based on the intergroup comparison of kappa coefficients for yearly and seasonal seafood consumption ( r  = 0.69 to 0.99)., Conclusions: The seafood FFQ instrument evaluated in this study, included as a supplement to this report, used in tandem with a photographic portion guide, provides a utilitarian tool for assessing fish, shrimp, oyster, and blue crab intake dynamics in adult and youth populations drawn from coastal communities., Competing Interests: The authors declare there are no conflicts of interest regarding the publication of this article., (Copyright © 2020 Makyba K. Charles-Ayinde et al.)