Advancement in next generation sequencing offers the possibility of routine use of whole genome sequencing (WGS) forMycobacterium bovis(M. bovis) genomes in clinical reference laboratories. To date, theM. bovisgenome could only be sequenced if the mycobacteria were cultured from tissue. This requirement for culture has been due to the overwhelmingly large amount of host DNA present when DNA is prepared directly from a granuloma. To overcome this formidable hurdle, we evaluated the usefulness of an RNA-based targeted enrichment method to sequenceM. bovisDNA directly from tissue samples without culture. Initial spiking experiments for method development were established by spiking DNA extracted from tissue samples with serially dilutedM. bovisBCG DNA at the following concentration range: 0.1 ng/μl to 0.1 pg/μl (10–1to 10–4). Library preparation, hybridization and enrichment was performed using SureSelect custom capture library RNA baits and the SureSelect XT HS2 target enrichment system for Illumina paired-end sequencing. The method validation was then assessed using direct WGS ofM. bovisDNA extracted from tissue samples from naturally (n= 6) and experimentally (n= 6) infected animals with variable Ct values. Direct WGS of spiked DNA samples achieved 99.1% mean genome coverage (mean depth of coverage: 108×) and 98.8% mean genome coverage (mean depth of coverage: 26.4×) for tissue samples spiked with BCG DNA at 10–1(mean Ct value: 20.3) and 10–2(mean Ct value: 23.4), respectively. TheM. bovisgenome from the experimentally and naturally infected tissue samples was successfully sequenced with a mean genome coverage of 99.56% and depth of genome coverage ranging from 9.2× to 72.1×. The spoligoyping andM. bovisgroup assignment derived from sequencing DNA directly from the infected tissue samples matched that of the cultured isolates from the same sample. Our results show that direct sequencing ofM. bovisDNA from tissue samples has the potential to provide accurate sequencing ofM. bovisgenomes significantly faster than WGS from cultures in research and diagnostic settings.