We developed and evaluated simple sequence repeat (SSR) markers derived from expressed sequence tags (ESTs) of Liriodendron tulipifera. Characteristics of 15 EST-SSR loci were investigated using 33 L. tulipifera individuals. The number of alleles per locus ranged from two to five. The expected and observed heterozygosities ranged from 0.216 to 0.751 and from 0.182 to 0.97, respectively. These loci were further tested for their cross-species transferability to Liriodendron Chinense. Because of their high level of polymorphism and transferability, our 15 single-locus EST-SSR markers will be valuable tools for research on mating system, population genetics and systemic evolution of Liriodendron.