Plant breeding that emphasizes crosses among elite parents in a closed population (advanced cycle breeding) is presumed to decrease genetic diversity. To assess the effect of plant breeding on allelic diversity, we evaluated regional ancestors, parental lines, and cultivar candidates from the University of Minnesota six-rowed barley (Hordeum vulgare L.) breeding program between 1958 and 1998 using pedigree information, 70 simple sequence repeat (SSR) markers, and a gene specific marker. Pedigree and SSR allelic diversity indices revealed a decrease in genetic diversity, from an average of 5.89 alleles per locus in the ancestors group to 2.34 alleles per locus in the fourth decade of breeding. A correspondence analysis showed differentiation in the germplasm with time. At specific loci, we detected both reductions and no change in the number of alleles over time. Several marker loci that demonstrated a reduction in number of alleles were associated with major loci for disease resistance or malting quality and were presumably under selection during breeding. Assessment of locus-specific allelic variation across the genome in breeding germplasm should identify both the regions of the genome that should be conserved and the regions of the genome where there are opportunities to introgress new allelic diversity without disrupting desirable gene complexes.