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Bovine chromosome 20 (BTA20) is associated with several quantitative trait loci (QTL) for meat tenderness, birth weight, milk yield and composition. Fine mapping of these QTL requires the development of additional informative markers to increase the resolution of the BTA20 genetic and physical maps. A BTA20-specific library was constructed by means of microdissection and microcloning, and screened for dinucleotide repeats with (CA)16 and (GT)16 oligos. A total of 60 new microsatellites (MS) were developed and characterized for polymorphism using the U.S. Department of Agriculture (USDA)/Meat Animal Research Center (MARC) bovine reference family, of which 53 markers were informative in this family. The number of alleles for these loci varied from 1 to 14, with an average of 6.5. Thirty-three of these MSs, together with 105 markers previously mapped to BTA20, were scored on a 7000-rad cattle-hamster whole-genome radiation hybrid panel (SUNbRH), resulting in a high-resolution RH7000 rad map for BTA20.