Technical Abstract: Cross-species DNA sequence comparison is the primary approach to discover regulatory elements by identifying highly conserved sequences due to evolutionary constraints. Previously, we reported that a systematic approach, combining position-specific weight matrixes (JASPAR) and phylogenetic footprinting algorithm (TFLOC), was implemented and optimized to identify transcription factor binding sites (TFBSs) in mammalian promoter regions within human-mouse-rat alignments. To generate a dataset of conserved TFBSs in the cattle genome, we further applied this approach to human-cattle-dog alignments. We first estimated the impact of evolutionary distance on predictive power. TFLOC and PhastCons were applied to various species combinations including human-chimpanzee-macaque, human-mouse-rat and human-cattle-dog. Computational prediction was compared with previously known sites at diverse genomic loci. Those newly discovered sites were further confirmed by experimental verifications including gel shifting and reporter assays. The best prediction was produced by the human-cattle-dog comparison with a higher sensitivity and a higher true-positive rate. The closer human-chimpanzee-macaque comparison produced more spurious sites, while the more distant human-mouse-rat comparison had a lower sensitivity. These results highlight the importance of choosing species at proper evolutionary distance for comparative genomics studies. The cattle TFBS dataset will be made freely available at the earliest opportunity to the scientific community through a public accessible website.