A high-resolution survey in the semi-enclosed Kalloni Gulf (Lesvos Island, NE Aegean Sea) revealed a plethora of oyster reef structures. The reefs have a relief of up to 6 m high and a length of 1.4 km at the deepest part (∼17 m) of the surveyed area and are developed in a fine-grained seabed. Acoustic backscatter variations caused by the presence and the type/distribution of different reef structures were mapped, resulting in the identification of four backscatter types. Sub-bottom acoustic information revealed the presence of similar buried features, which were classified in three types according to their characteristics. The seismic stratigraphy also showed the Holocene highstand and the transgressive system tracts as well as a deeper-seated distinct erosional surface with numerous buried channels, most likely corresponding to the surface flooded during the Holocene marine transgression. Starting at around 9700 years BP, the Kalloni paleo-landscape was progressively submerged and gradually transformed into a shallow, land-locked marine environment, allowing the establishment, and thriving of oyster reefs. The buried reefs presumably began to grow during the early stages of the gulf's transgression, whereas most of the modern reefs were developed/maintained by the modern hydrodynamic/sedimentary regime. It is the first time that such a large occurrence of well developed, diachronic oyster reef assemblage has been documented in the transgressive coastal environments of the microtidal Mediterranean Sea, but further research is required to determine the exact mode of their development.