Although Japanese morning glory (Ipomoea nil (L.) Roth.) has been used intensively for genetic studies, DNA markers have not been developed in Ipomoea nil sufficient to cover all chromosomes. Therefore, we conducted microsatellite (simple sequence repeats, SSR) marker development in I. nil for future genetic studies. From 92,662 expressed sequence tag (EST) sequences, 514 unique microsatellite-containing ESTs were identified. Primer pairs were designed automatically in 326 SSRs. Of 150 SSRs examined, 75 showed polymorphisms among strains. A phenogram based on the SSR genotypes revealed the genetic relation among seven Japanese morning glories from five different regions of the world and an ivyleaf morning glory (I. hederacea Jacq.). The developed SSR markers might be applicable for genetic studies of morning glories and their relatives.