The fan mussel, Pinna nobilis is a highly endangered bivalve species endemic to the Mediterranean Sea. During the last few decades, populations have been greatly reduced due to anthropic impacts and they are now under strict protection in most Mediterranean countries. Today, the species is facing a major crisis following the introduction of an haplosporidan parasite which is driving mass mortality in almost all P. nobilis populations throughout the Mediterranean Sea. Gathering additional knowledge regarding dynamics and connectivity patterns of P. nobilis populations is now more than ever critical. Here, we describe the development of 26 highly polymorphic microsatellite markers. Average allelic diversity of 10.9 alleles per locus was reported and heterozygosity ranged from 0.0294 to 0.9737. We tested cross-species amplification in four Pinna species for the new markers together with 10 already published markers, and analysed its success according to the genetic distances among species. Cross-species transferability success ranged from 3 to 38% and had a negative relationship with the genetic distance between the target species and the tested species. The establishment of this new set of high-resolution markers provides a useful tool to understand processes driving gene flow and genetic diversity in P. nobilis populations and the closest congeneric species.