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Species belonging to the Mycobacterium tuberculosis Complex (MTBC) show more than 99% genetic identity but exhibit distinct host preference and virulence. The molecular genetic changes that underly host specificity and infection phenotype within MTBC members have not been fully elucidated. Here, we analysed RD900 genomic region across MTBC members using whole genome sequences from 60 different MTBC strains so as to determine its role in the context of MTBC evolutionary history. The RD900 region comprises two homologous genes, pknH1 and pknH2, encoding a serine/threonine protein kinase PknH flanking the tbd2 gene. Our analysis revealed that RD900 has been independently lost in different MTBC lineages and different strains, resulting in the generation of a single pknH gene. Importantly, all the analysed M. bovis and M. caprae strains carry a conserved deletion within a proline rich-region of pknH, independent of the presence or absence of RD900. We hypothesized that deletion of pknH proline rich-region in M. bovis may affect PknH function, having a potential role in its virulence and evolutionary adaptation. To explore this hypothesis, we constructed two M. bovis 'knock-in' strains containing the M. tuberculosis pknH gene. Evaluation of their virulence phenotype in mice revealed a reduced virulence of both M. bovis knock-in strains compared to the wild type, suggesting that PknH plays an important role in the differential virulence phenotype of M. bovis vs M. tuberculosis. Author summary: Tuberculosis is caused in humans and animals by organisms from the Mycobacterium tuberculosis Complex (MTBC), that share more than 99% genetic identity but exhibit distinct host preference and virulence. While Mycobacterium tuberculosis is the main causative agent of human TB, Mycobacterium bovis is responsible for bovine TB disease, that exacts a tremendous economic burden worldwide, as well as being a zoonotic threat. Unlike the human restriction of M. tuberculosis, M. bovis has a broader host range and it has been found to be more virulent than M. tuberculosis in different animal models. However, the molecular basis for host preference and virulence divergence between M. tuberculosis and M. bovis is not fully elucidated. Here we study the genetic variations of the genomic region RD900 in the context of MTBC phylogeny. RD900 contains two genes encoding orthologues of the serine/threonine kinase PknH, which is linked to the regulation of several bacterial processes including virulence. We found that M. bovis pknH genes show a conserved deletion that is not present in M. tuberculosis strains, and we evaluated the potential impact of these variations in the regulation of M. bovis vs M. tuberculosis virulence through the construction and in vivo characterization of M. bovis pknH mutant strains. [ABSTRACT FROM AUTHOR]