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Premise of the study: We developed microsatellite primers to investigate genetic diversity and population genetic structure of the cosmopolitan submerged plant <i>Myriophyllum spicatum</i>. Methods and Results: Twenty microsatellite loci were identified in <i>M. spicatum</i> using the microsatellite-enriched library method. The numbers of alleles per locus ranged from one to 13, and the expected heterozygosity varied from 0 to 0.873 with a mean of 0.504 in two Chinese populations of <i>M. spicatum</i>. All of the loci were also found to be amplifiable in the related species <i>M. verticillatum</i> and <i>M. sibiricum</i>. Conclusions: The results indicate that these markers will be significant for studies of population genetic structure and evolutionary history of <i>M. spicatum</i> as well as some of its related species.