The expressed sequence tag (EST) database represents a potentially valuable resource for the development of simple sequence repeat (SSR) markers for use in evolutionary studies. EST-SSRs reveal polymorphisms not only within the source taxon, but in related taxa as well. In this paper, we describe a case study in which the publicly available walnut (Juglans regia) EST database was used to develop SSR markers for use in the genetic analysis of the widespread Juglans nigra and Carya cathayensis and an endangered species Annamocarya sinensis. A total of 7262 unigenes, including 1911 contigs and 5351 singletons, were obtained from 13,559 ESTs retrieved from the NCBI database. The 7262 unigenes were further reduced to 706 EST-SSR sequences containing 805 SSR loci. Then, 309 EST-SSR primers were randomly designed, and 77 were identified with five high across-species transferability cross-species: namely, J. regia, J. nigra, C. cathayensis, Carya dabieshanensis, and A. sinensis. Thirteen highly polymorphic EST-SSRs were further used for genetic analyses in these above five species.