Abstract Expressed sequence tags (EST), which have been increasing rapidly in number recently, provide important resources for the development of molecular markers. Compared with conventional markers derived from genomic DNA, the EST-derived markers are a novel type of molecular tool with remarkable advantages such as being easy to develop, more informative, and highly transferable. EST markers have been used in many research fields. In the present paper, five kinds of recently developed EST markers, including EST-PCR, EST-SSR, EST-SNP, EST-RFLP, and EST-AFLP, as well as their development strategies based on bioinformatics, are introduced. Moreover, the applications of these markers in comparative genomics studies, including comparative mapping, genetic diversity evaluation, and phylogenetics and so on, are also reviewed.