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Background Bovine tuberculosis has led to serious economic losses for Sika Deer producers in China. Strategies for controlling the spread of Mycobacterium bovis are often hampered by a lack of epidemiological data. Specifically, tracing infections requires the ability to trace back infections, which, in turn, requires the ability to determine isolates with a common source. This study was planned to assess the discriminatory power of each mycobacterial interspersed repetitive unit (MIRU)-variable number tandem repeats (VNTR) locus and evaluate the most appropriate combination of MIRU-VNTR loci for molecular epidemiological studies on Sika Deer in China. Results The discriminatory power of MIRU-VNTR typing based on 22 known loci (12 MIRUs, 2 ETRs, 4 QUBs, and 4 Mtubs) were assessed in 96 Mycobacterium bovis strains collected sequentially from Sika Deer at a slaughterhouse in northeastern China. We defined four loci (MIRU4, ETRA, QUB11b, and Mtub4) as highly discriminative, eight loci (MIRU2, MIRU23, MIRU27, MIRU31, MIRU39, MIRU40, QUB26, and Mtub21) as moderately discriminative, and three loci (MIRU16, Mtub30, and Mtub34) as poorly discriminative. The final locus showed no polymorphism between strains. MIRU-VNTR typing as a whole was highly discriminative, with an overall allelic diversity of 0.897. Of the loci tested, the four highly discriminative loci and eight moderately discriminative loci proved to be most appropriate for first line typing of M. bovis from Sika Deer, with the same resolving ability as all 22 loci (H = 0.897). Conclusions MIRU-VNTR typing is quick and effective for typing bovine tuberculosis isolates from Sika Deer in China. Electronic supplementary material The online version of this article (doi:10.1186/s12917-015-0402-0) contains supplementary material, which is available to authorized users.