The cattle chromosome region BTA5q21-q25 is associated with immune defense and is linked with several economically important traits for growth, ovulation, twinning and milk yield. Chromosome microdissection of the region was performed to generate fragment-specific DNA libraries and to provide new bovine-specific DNA sequences as starting material for physical fine-mapping approaches or linkage studies. A first library screening resulted in the identification of 16 region-specific sequence-tagged sites (STS). Fifteen STS represent new unique cattle DNA sequences, of which one identifies a new microsatellite. Homology with the gene CDK2 was found for the remaining STS.