• Premise of the study: A total of 17 polymorphic microsatellite markers were developed for the tropical Atlantic seagrass Syringodium filiforme (Cymodoceaceae), enabling analysis of population genetic structure in this species for the first time.• Methods and Results: The 17 primers amplified di‐ and trinucleotide repeats revealing two to eight alleles per locus among the South Florida populations tested. In the analysis of two populations from the Florida Keys (Florida, USA), observed heterozygosity ranged from 0.063 to 0.905, although sampling was from relatively closely located populations so heterozygosity is expected to be higher across larger spatial scales. Multiplex PCRs consisting of two 6‐plex and one 5‐plex reactions were developed to maximize genotyping efficiency.• Conclusions: We present here 17 polymorphic markers that will be useful for the study of clonality and population structure of S. filiforme, a marine plant that forms extensive habitat throughout the tropical Atlantic and Caribbean.