Ten new microsatellite markers were isolated from the freshwater blenny, Salaria fluviatilis. These loci were characterised for three distinct populations (Var River and Lake Bourget in France, and Lake Kinneret in Israel). S. fluviatilis populations in the North-West of the Mediterranean Sea are all polymorphic and under Hardy– Weinberg equilibrium. These microsatellite markers are suggested for studying the genetic structure in this area. Seven of the ten microsatellite markers are applicable for all the freshwater blenny populations of the entire Mediterranean basin. Cross-species characterisation was also performed on the peacock blenny, Salaria pavo. Seven of the ten microsatellite loci exhibited successful PCR amplification on this latter species. Polymorphism is observed on four of these seven loci.