Mycobacterium simiae (Karassova V, Weissfeiler J, Kraszanay E, Acta Microbiol Acad Sci Hung 12:275-82, 1965) is a slow-growing nontuberculous Mycobacterium species found in environmental niches, and recently evidenced as an opportunistic Human pathogen. We report here the genome of a clinical isolate of M. simiae (MsiGto) obtained from a patient in Guanajuato, Mexico. With a size of 6,684,413 bp, the genomic sequence of strain MsiGto is the largest of the three M. simiae genomes reported to date. Gene prediction revealed 6409 CDSs in total, including 6354 protein-coding genes and 52 RNA genes. Comparative genomic analysis identified shared features between strain MsiGto and the other two reported M. simiae genomes, as well as unique genes. Our data reveals that M. simiae MsiGto harbors virulence-related genes, such as arcD, ESAT-6, and those belonging to the antigen 85 complex and mce clusters, which may explain its successful transition to the human host. We expect the genome information of strain MsiGto will provide a better understanding of infective mechanisms and virulence of this emergent pathogen.