© 2015 Enrique Bolado-Martínez et al. Mycobacterium bovis is the major causative agent of bovine tuberculosis, one of the most relevant zoonoses in the world, and affects a wide range of wild and domesticated animals. Development of screening panels in mycobacterial genotyping, according to specific geographical regions, is strongly needed. The aim of this study is to select a panel, constituted by highly polymorphic MIRU-VNTR loci, to discriminate clinical isolates of M. bovis in Mexico. In this study, 65 isolates of M. bovis obtained from clinical bovine samples proceeding from different geographic regions of Mexico were identified by phenotypic and genotypic tests and subsequently genotyped by a 24-locus MIRU-VNTR panel. The most polymorphic loci were selected to build a panel with a high discriminatory power similar to the 24-locus panel results. A panel of seven elements (QUB 11a, MIRU 26, ETR-A, QUB 26, MIRU 16, MIRU 27, and MIRU 39) with the highest allelic diversity showed an appropriate differentiation. The selected MIRU-VNTR elements, according to the regional allelic variability, may be used in the preliminary genotyping of Mycobacterium bovis isolates in Mexico.