• Premise of the study: To capture molecular markers that are applicable to environmental risk assessment of genetically modified oilseed rape, and to streamline their development, we screened variations in nucleotide sequences of three Brassica species by DNA microarray analysis.• Methods and Results: Using the Affymetrix GeneChip Arabidopsis ATH1 Genome Array, we monitored gene expression at 22810 loci among the Brassica species and picked out 192 putative polymorphic loci. We sequenced 25 of these and successfully aligned them among all three species. All 25 loci possessed some interspecific and at times intraspecific nucleotide variation.• Conclusions: DNA microarray analysis effectively detected a large number of nucleotide sequence variations among closely related Brassica species. The polymorphic regions will allow the subsequent development of functional gene markers.