The discriminatory power of variable number tandem repeat (VNTR) typing based on 16 known loci (12 MIRUs, 3 ETRs and VNTR 3232) was assessed for Mycobacterium bovis strains collected sequentially at the slaughterhouse of N'Djaména, Chad. Of 67 M. bovis strains analyzed, 67% were clustered. In this study, VNTR typing was highly discriminative with an overall allelic diversity (h(oa)) of 0.922. We defined five loci (ETR A, B, C and MIRU 26, 27) as highly (h>0.25), two loci (MIRU 4, and VNTR 3232) as moderately (0.11<h<0.25) and three loci (MIRU 16, 20, 31) as poorly (0.01<h<0.11) discriminative. Six loci (MIRU 2, 10, 23, 24, 39, and 40) showed no polymorphism at all. VNTR typing of the five highly discriminative loci (h=0.917) proved to be most appropriate for first line typing of M. bovis strains of Chad and superior than spoligotyping (h(sp)=0.789). In contrast to Mycobacterium tuberculosis strains, a consensus on VNTR loci needs to be found for M. bovis strains. The selection of a generally agreed set of VNTR loci for molecular discrimination of M. bovis in different geographical settings is discussed.