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Caseous lymphadenitis (CLA) is a worldwide disease of small ruminants caused by <i>Corynebacterium pseudotuberculosis</i>, a facultative intracellular pathogen that is able to survive and multiply in certain white blood cells of the host. In this study, 33 strains of <i>C. pseudotuberculosis</i> were isolated from sheep and goats suffering from CLA on nine farms in the Czech Republic. All these strains were tested for their antibiotic susceptibility, ability to form a biofilm and resistance to the effects of commonly used disinfectant agents. To better understand the virulence of <i>C. pseudotuberculosis</i>, the genomes of strains were sequenced and comparative genomic analysis was performed with another 123 genomes of the same species, including <i>ovis</i> and <i>equi</i> biovars, downloaded from the NCBI. The genetic determinants for the virulence factors responsible for adherence and virulence factors specialized for iron uptake and exotoxin phospholipase D were revealed in every analyzed genome. Carbohydrate-Active Enzymes were compared, revealing the presence of genetic determinants encoding exo-α-sialidase (GH33) and the CP40 protein in most of the analyzed genomes. Thirty-three Czech strains of <i>C. pseudotuberculosis</i> were identified as the biovar <i>ovis</i> on the basis of comparative genome analysis. All the compared genomes of the biovar <i>ovis</i> strains were highly similar regardless of their country of origin or host, reflecting their clonal behavior.