Mycobacteroides immunogenum is an emerging opportunistic pathogen implicated in nosocomial infections. Comparative genome analyses may provide better insights into its genomic structure, functions and evolution. The present analysis showed that M. immunogenum has an open pan-genome. Approximately 36.8% of putative virulence genes were identified in the accessory regions of M. immunogenum. Phylogenetic analyses revealed two potential novel subspecies of M. immunogenum, supported by evidence from ANIb (average nucleotide identity using blast) and GGDC (Genome to Genome Distance Calculator) analyses. We identified 74 genomic islands (GIs) in Subspecies 1 and 23 GIs in Subspecies 2. All Subspecies 2-harboured GIs were not found in Subspecies 1, indicating that they might have been acquired by Subspecies 2 after their divergence. Subspecies 2 has more defence genes than Subspecies 1, suggesting that it might be more resistant to the insertion of foreign DNA and probably explaining why Subspecies 2 has fewer GIs. Positive selection analysis suggest that M. immunogenum has a lower selection pressure compared to non-pathogenic mycobacteria. Thirteen genes were positively selected and many were involved in virulence.