Technical Abstract: DNA marker technology represents a promising means for determining the genetic identity and kinship of an animal. Compared to other types of DNA markers, single nucleotide polymorphisms (SNPs) are attractive because they are abundant, genetically stable, and amenable to high-throughput automated analysis. In cattle, the challenge has been to identify a minimal set of SNPs with sufficient power for use in a variety of popular breeds and crossbred populations. This report describes a set of 32 highly informative SNP markers distributed among 18 autosomes and both sex chromosomes. Informativity of these SNPs in U.S. beef cattle populations was estimated from the distribution of allele and genotype frequencies in two panels: one consisting of 96 purebred sires representing 17 popular breeds and another with 154 purebred American Angus from six herds in four Midwestern states. Based on frequency data from these panels, estimated probability that two randomly selected, unrelated individuals will posses identical genotypes for all 32 loci was 2.0 x 10^-13 for multi-breed composite populations and 1.9 x 10^-10 for purebred Angus populations. The probability that a randomly chosen candidate sire will be excluded from paternity was estimated to be 99.9% and 99.4% for the same respective populations. The DNA immediately surrounding the 32 target SNPs was sequenced in the 96 sires of the multi-breed panel and found to contain an additional 183 polymorphic sites. Knowledge of these additional sites, together with the 32 target SNPs, allows the design of robust accurate genotype assays on a variety of high-throughput SNP genotyping platforms.