Mycobacterium microti is a member of the Mycobacterium tuberculosis complex of bacteria. This species was originally identified as a pathogen of small rodents and shrews and was associated with limited diversity and a much reduced spoligotype pattern. More recently, specific deletions of chromosomal DNA have been shown to define this group of organisms, which can be identified by the absence of chromosomal region RD1mic. We describe here the molecular characteristics of 141 strains of the Mycobacterium tuberculosis complex isolated in Great Britain over a 14-year period. All strains have characteristic loss of some spoligotype spacers and characteristic alleles at the ETR-E and ETR-F variable-number tandem-repeat (VNTR) loci, and a sample of these strains was deleted for regions RD7, RD9, and RD1mic but intact for regions RD4 and RD12. We therefore identified these strains as M. microti and show that they have much more diverse spoligotype patterns and VNTR types than previously thought. The most common source of these strains was domestic cats, and we show that the molecular types of M. microti are geographically localized in the same way that molecular types of Mycobacterium bovis are geographically localized in cattle in the United Kingdom. We describe the pathology of M. microti infection in cats and suggest that the feline disease is a spillover from a disease maintained in an unknown wild mammal, probably field voles. The location of the cats with M. microti infection suggests that they do not overlap geographically with the strains of Mycobacterium bovis in Great Britain.