We report AtPRIMER, an application that automates the discovery of new polymorphic markers between ecotypes of Arabidopsis thaliana. On specifying two ecotypes and the genomic region of interest, the script retrieves all corresponding single nucleotide polymorphisms (SNPs) and generates CAPS and/or dCAPS PCR primer sequences. We show that AtPRIMER accurately found specific polymorphic markers for our linkage mapping project. AtPRIMER will therefore be useful for efficient marker development with high density and specificity.