Objective To obtain microsatellite DNA sequence of Microtus fortis from its BAC library. MethodsTo screen the microsatellite DNA sequence with nonradioactive analysis method—colony hybridization and enrichments by magnetic beads. ResultsTwenty positive clones with the strongest signals were screened out of 136 BAC clones through hybridization of digoxin-labeled oligonucleotide (CA)₂₀. Then these positive BAC clones were devoted to construct the subclone libraries separately. 220 microsatellite sequences were isolated from 400 positive subclones identified by PCR. According to those microsatellite sequences with more copy numbers and complete flanking sequence,74 pairs of primers were designed. As a result, 35 pairs of microsatellite primers got clear bands, of which 16 pairs had polymorphism. ConclusionsMicrosatellite DNA sequences have been screened successfully and efficiently from BAC library of Microtus fortis. The sequences and positive BAC clones may contribute to further research of their location. 