Members of the Mycobacterium tuberculosis complex (MTC) are causative agents of tuberculosis (TB) in both humans and animals. MTC species are genetically very similar but may differ in their epidemiology, namely geographic distribution and host preferences, virulence traits and antimicrobial susceptibility patterns. However, the conventional laboratory diagnosis does not routinely differentiate between the species of the MTC. In this work we describe a rapid and robust two-step five-target probe-based real-time PCR identification algorithm, based on genomic deletion analysis, to identify the MTC species most commonly associated with TB in livestock and other animals. The first step allows the confirmation of the cultures as MTC members, by targeting their IS6110 element, or as a mycobacterial species, if only a 16S rDNA product is detected in the duplex amplification reaction. If a MTC member is identified, the second amplification step allows to assess the presence or absence of the RD1, RD4 and RD9 genomic regions. The correspondent pattern allows to infer the species of the isolate as M. tuberculosis (if all RDs are present), M. caprae (if only RD1 and RD4 are present) and M. bovis (if only RD1 is present). The identification algorithm developed presented an almost perfect agreement with the results of the routine bacteriological analysis, with a kappa coefficient of 0.970 (CIP95% 0.929-1.000). The assay is able to be adaptable to automation and implementation in the routine diagnostics framework of veterinary diagnostics laboratories, with a particular focus for reference laboratories.