To further the understanding of the evolution of transcriptional regulation, we profiled genome-wide transcriptional start sites (TSSs) in two sub-species, Bos taurus taurus and Bos taurus indicus, that diverged approximately 500,000 years ago. Evolutionary and developmental-stage differences in TSSs were detected across the sub-species, including translocation of dominant TSS and changes in TSS distribution. The 16% of all SNPs located in significant differentially used TSS clusters across sub-species had significant shifts in allele frequency (472 SNPs), indicating they may have been subject to selection. In spleen and muscle, a higher relative TSS expression was observed in Bos indicus than Bos taurus for all heat shock protein genes, which may be responsible for the tropical adaptation of Bos indicus.