Sixty-nine sequences containing microsatellites were determined by analysis of clones from a pUC118 library of total genomic mouse DNA. These sequences were examined for size variation using polymerase chain reaction and gel electrophoresis. Fifty-one of them showed allelic variations between C57BL/6 and MSM, the two strains used for genetic mapping. Hence, their chromosomal location was determined using a panel consisting of 131 backcross mice that had been typed with 85 anchor loci. The microsatellites were distributed to most chromosomes except for chromosomes 16 and 19. These novel markers with defined locations are useful in linkage and genome mapping studies.