The availability of expressed sequence data derived from gene discovery programs enables mining for simple sequence repeats (SSR), providing useful genetic markers for crop improvement. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study presents the development and characterization of 24 expressed sequence tags (EST)-SSR markers from Brassica napus and their cross-amplification across Brassica species. The markers show reliable amplification, genome specificity and considerable polymorphism, demonstrating the utility of EST-SSRs for genetic analysis of wild Brassica populations and commercial Brassica germplasm.