The lack of a convenient high-resolution strain-typing method has hampered the application of molecular epidemiology to the surveillance of bacteria of the Mycobacterium tuberculosis complex, particularly the monitoring of strains of Mycobacterium bovis. With the recent availability of genome sequences for strains of the M. tuberculosis complex, novel PCR-based M. tuberculosis-typing methods have been developed, which target the variable-number tandem repeats (VNTRs) of minisatellite-like mycobacterial interspersed repetitive units (MIRUs), or exact tandem repeats (ETRs). This paper describes the identification of seven VNTR loci in M. tuberculosis H37Rv, the copy number of which varies in other strains of the M. tuberculosis complex. Six of these VNTRs were applied to a panel of 100 different M. bovis isolates, and their discrimination and correlation with spoligotyping and an established set of ETRs were assessed. The number of alleles varied from three to seven at the novel VNTR loci, which differed markedly in their discrimination index. There was positive correlation between spoligotyping, ETR- and VNTR-typing. VNTR-PCR discriminates well between M. bovis strains. Thirty-three allele profiles were identified by the novel VNTRs, 22 for the ETRs and 29 for spoligotyping. When VNTR- and ETR-typing results were combined, a total of 51 different profiles were identified. Digital nomenclature and databasing were intuitive. VNTRs were located both in intergenic regions and annotated ORFs, including PPE (novel glycine-asparigine-rich) proteins, a proposed source of antigenic variation, where VNTRs potentially code repeating amino acid motifs. VNTR-PCR is a valuable tool for strain typing and for the study of the global molecular epidemiology of the M. tuberculosis complex. The novel VNTR targets identified in this study should additionally increase the power of this approach.