ABSTRACT: The present study examines the genetic diversity (using cytochrome c oxidase subunit I [COI] haplotypes) of polychaete Hediste diversicolor populations in the Amvrakikos Gulf (western Greece) in order to test Cognetti’s hypothesis on the formation of distinct isolated populations of species living in stressed transitional water habitats. To this purpose, the intraspecific diversity of H. diversicolor and the similarities among its populations in the brackish water habitats of the Eastern Mediterranean were investigated. Samples were collected from the lagoonal complex of Amvrakikos Gulf in 2010 and 2011. Analysis showed that populations in the Amvrakikos Gulf share unique haplotypes which are distinct from the haplotypes hitherto reported, suggesting that these populations are highly isolated. This evidence partially supports Cognetti’s hypothesis. The results indicate that the genetic differentiation in the studied populations was probably caused by the complete isolation of the gulf and periodic hypoxic conditions during its formation history. The occurrence of unique haplotypes in the gulf underlines the importance of considering genetic diversity in the design of long-term biodiversity management and conservation plans.