We recently proposed a new PCR-based genetic marker assay for the mouse genome that exploits sequence differences in the 3'-untranslated region (UTR) of cDNAs between different mouse strains, called "biallelic polymorphic expressed sequence tags (bESTs)." The specific use of 3'-UTR has several advantages: (1) frequent sequence polymorphism between different mouse strains, (2) most commonly uninterrupted by introns, (3) usually unique sequence even among closely related gene family members. In this paper, we identify additional genetic loci defined by bEST and determine their location on the mouse genetic map by using interspecific backcross mapping panels between C57BL/6J and Mus spretus. Of 136 markers tested, 86 produced unique PCR products from C57BL/6J and M. spretus genomic DNAs. We then sequenced these 86 PCR products from C57BL/6J and M. spretus and found that 59 markers have sequence polymorphisms. Of these, we mapped 36 by restriction fragment length polymorphism (RFLP) of the PCR products and 4 by length polymorphism (LP) of the PCR products. We discuss the possibility of a large-scale application of this method for cDNA mapping.