Background <p><i>Corynebacterium pseudotuberculosis</i>, a gram-positive bacterium, causes caseous lymphadenitis in small ruminants (biovar ovis) and ulcerative lymphangitis in equines (biovar equi). Growing reports of <i>C. pseudotuberculosis</i> from various animals, including cattle, buffalo, pigs, deer, Iberian ibex, camel, llama, and alpaca. Since the host range is expanding, there is a need for biovar-specific identification of the organism. Hence, the present study was focused towards comparative genomics of <i>C. pseudotuberculosis</i> to identify biovar ovis specific markers.</p> Methods and results <p><i>Corynebacterium&#xa0;pseudotuberculosis</i> TN_CUL_1 was isolated from suspected cases of caseous lymphadenitis from small ruminants in an organized farm in Tamil Nadu, India. The <i>C. pseudotuberculosis</i> TN_CUL_1 strain was whole genome sequenced and was used for comparative genome analysis along with 157 <i>C. pseudotuberculosis</i> strains available in the NCBI genome database. Whole genome phylogeny showed a clear distinction between biovars equi and ovis, and the TN_CUL_1 strain was clustering in biovar ovis. Virulence gene <i>pld</i> was predicted in all the strains, while 9 strains isolated from buffalo had the <i>tox</i> gene coding for diphtheria toxin. Pangenome analysis indicated that the genome structure of <i>C. pseudotuberculosis</i> to be closed in nature as the pangenome curve attained a plateau. Biovar-specific genes like <i>narT</i>,<i> narK</i>,<i> narY</i>,<i> narG</i>,<i> narX</i>, <i>moaC</i>,<i> moaA</i>,<i> mobA</i>,<i> and moeZ</i> were predicted only in biovar equi, while <i>yhaP</i> was predicted only in biovar ovis strains. Further BLAST analysis indicated that the <i>yhap</i> gene coding for the ABC superfamily ATP binding cassette transporter binding protein was found only in biovar ovis strains.</p> Conclusions <p>The study concludes that the <i>yhap</i> gene can be used as a biovar ovis-specific molecular marker.</p>