The Manila clam (Ruditapes philippinarum) is one of the most traded bivalves in the world. Knowing its harvesting location is therefore paramount to guarantee the safety of consumers. The present study employs fatty acid (FA) profiles of the adductor muscle (AM) to reveal the most likely harvesting location of four batches of Manila clams suspected of having been illegally sourced from the Tagus estuary. In this ecosystem, where the collection of Manila clams is currently prohibited for food safety reasons, illegal, unreported and unregulated (IUU) capture is known to occur. In order to trace the geographic origin of these four batches of Manila clams, a reference model based on the FA profiles of the AM was developed with specimens originating from the two most representative ecosystems supplying the trade-chain of this species in mainland Portugal (the Tagus estuary and Ria de Aveiro), as well as Ría de Vigo, a production area outside Portugal and that is also an important supplier. The ability of this model to allocate clams to its origin ecosystem was evaluated using independent datasets, with an allocation success of 100% (all samples were correctly assigned to its origin ecosystem, thus validating the model). Based on the reference model established, the harvesting location of the four batches suspected of originating from ongoing IUU in the Tagus estuary was investigated. Specimens from 3 of the 4 batches screened were classified, as most likely originating from the Tagus estuary (with a likelihood ranging from 90% up to 100%). These results highlight the potential of this approach to fight the IUU capture of Manila clams, as this practice endangers important habitats and threatens public health.