Abstract: The aim of present study was to reveal the comparative differences of whole-genome sequences between Mycobacterium tuberculosis and Mycobacterium bovis, and to provide new molecular markers for identification and diagnosis of these two bacilli. The whole-genome sequences of M. tuberculosis H37Rv and M. bovis AF2122/97 were sequenced and their genomic comparison was performed by on-line tools. M. bovis AF2122/97 shared 99.95% genomic nucleotides identity with M. tuberculosis H37Rv. 14 large fragment deletions varied in a range from 0.8 to 12.7 kb and 6 small deletions varied from 12 to 714 bp were detected in AF2122/97 strain by comparison with H37Rv, while 6 large deletions ranged from 1.3 to 5.4 kb and 5 small deletions ranged from 10 to 48 bp, in turn, were discovered in H37Rv strain. The large fragment deletions of RD18, RD19 and RD20 in AF2122/97 strain, and all small deletions both in AF2122/97 and H37Rv strains were firstly reported. Deletion has led to a reduced genome size of M. bovis. Most of the deleted genes are found to be responsible for encoding cell wall components and secreted proteins, indicating that deletions of these genes may contribute to hostbacillus interaction or immune evasion. The new understanding might be helpful in the development of new drugs, diagnostic reagents and vaccines.