We have developed 13 microsatellite loci from an enrichment library of genomic DNA in the evergreen shrub Ilex leucoclada. One hundred and eighty-nine out of 432 clones were found to contain microsatellite repeats. Primer pairs were designed for 92 of these clones according to their sequence data. Thirteen of these primer pairs revealed polymorphism among 36 individuals sampled from 12 populations. Three to 27 alleles per locus were detected, and the expected heterozygosity ranged from 0.133 to 0.971. Because these 13 microsatellite markers showed high degrees of genetic variation, they should be useful tools for studying population and ecological genetics of I. leucoclada.