Effective animal identification systems benefit agriculture by facilitating efficient responses to disease outbreaks. However, even in areas where animal identification systems are operational, correctly tracing the source of an outbreak is difficult and conclusions may be disputed. DNA fingerprinting (i.e., comparing genotypes between samples) resolves disputes if samples were collected at the point of origin before a disease outbreak occurred. In the absence of pre-existing samples, parentage testing, (i.e., determining whether alleles are shared between parents and offspring) may identify the origin of a diseased animal if tissues from a parent are available. Our aim was to develop DNA markers with maximum power for parentage-based DNA traceback in U.S. and Canadian cattle and amenability to rapid, high-throughput genotyping. Single nucleotide polymorphism (SNP) markers were screened for allele frequency and genome-wide distribution. A set of more than 100 SNPs with an average minor allele frequency greater than 0.41 was identified in a group of 216 diverse sires from 19 beef and 4 dairy breeds. The average probability of excluding an unrelated sire with these markers was greater than 0.99999 with no dam available. The 1000-bp region surrounding these SNPs was sequenced in the same group of 216 sires to document more than 1400 additional polymorphisms. Knowledge of this flanking nucleotide diversity substantially increases the overall accuracy of DNA tests. This set of markers is being developed for multiple DNA diagnostic uses and is publicly available for use by researchers, producers, forensic specialists, commercial genotyping laboratories, and regulatory agencies.