The kelp Saccharina latissima is a species of high ecological and economic importance. We developed a novel set of S. latissima-specific genetic markers that will find applications in conservation biology, biodiversity assessment, and commercial exploitation of this macroalga. Thirty-two expressed sequence tag (EST)-derived microsatellite markers (SSRs) were developed and characterized in this study using publically available EST sequences. Twenty-seven percent of the 7064 analyzed ESTs contained repeat motifs, and polymerase chain reaction (PCR) amplification primers were designed for 96 selected loci. Fifty-one (53 %) of the primer pairs amplified their target loci, of which 32 (33 %) were polymorphic within a sample of 96 S. latissima sporophytes collected from six localities distributed along the European Atlantic coast from Southern Brittany (France) to Spitzbergen (Norway). The 32 loci harbored moderate levels of polymorphism with 2–13 alleles per locus (mean 5.4). The 25 loci that were retained for population genetic analyses revealed substantial genetic differentiation among the European populations (pairwise F ST values ranging from 0.077 to 0.562) that did not follow any pattern of isolation by distance. In addition, within-population genetic diversity was generally low (Hs < 0.323). Two non-mutually exclusive hypotheses were proposed to explain this low diversity pattern: (1) lower variability of the EST-derived microsatellites compared to the random distribution of SSRs developed from genomic DNA since the former are frequently located in coding regions, which are generally less variable, or (2) reduced effective population size of S. latissima. The particularly high genetic differentiation between the French and Scandinavian S. latissima populations is in agreement with the reported ecotypic differentiation, which may reflect an important resource for genetic improvement. The pattern of genetic diversity revealed in this study thus suggests that care should be taken to avoid the transfer of strains between different geographic regions.