Abstract Summary: 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. Availability: This PERL/CGI program is available for non-commercial users at http://www.AtPRIMER.tsl.ac.uk. The web-based version is available for public use at the same URL. Contact: [email protected]