<p>Tidal flats are dynamic ecosystems where bivalves play a key role in nutrient cycling through selective feeding on suspended and benthic organic matter. However, conventional bulk stable isotope analysis can have limited resolving power for distinguishing among multiple food sources when their carbon isotope ratios (δ¹³C) overlap, particularly with respect to identifying the dominant organic matter sources. In this study, we evaluated the applicability of fatty acid compound-specific isotope analysis (FA-CSIA) to identify the food sources of benthic organisms inhabiting tidal flat ecosystems. We collected bivalves and polychaetes from the Gamou tidal flat and analyzed their δ¹³C values using bulk isotope analysis, fatty acid biomarkers, and FA-CSIA. Principal component analysis of fatty acid biomarkers revealed differences in the characteristics of benthic organisms and their food sources. FA-CSIA improved the accuracy of source identification for essential fatty acids such as linoleic acid and α-linolenic acid. Bivalves showed isotopic signatures similar to those of particulate organic matter (POM), while polychaetes aligned more closely with those of sediment organic matter (SOM). These findings suggest that FA-CSIA can effectively resolve trophic relationships in complex environments where conventional methods are limited.</p>