Caseous lymphadenitis is a highly prevalent worldwide disease of sheep and goats caused by C. pseudotuberculosis, resulting in significant economic losses. In order to investigate the prevalence as well as the molecular characterization of C. pseudotuberculosis and monitoring the antibiotic susceptibility of the isolated strains, a total of 126 pus samples were collected aseptically from 80 sheep and 46 goats suffering from abscessation. The collected samples were subjected to bacteriological examination where the prevalence of C. pseudotuberculosis was 43.8% and 36.9% in diseased sheep and goats, respectively. The antibiotic sensitivity test was carried out using disc diffusion method, most of the isolates were resistant to penicillin (96.2%) and erythromycine (92.3%), highly sensitive to ciprofloxacin (96.2%), amikacin (90.4%) and neomycin (88.5%). Polymerase chain reaction was applied on 30 C. pseudotuberculosis isolates for the amplification and detection of both 16S rRNA and the RNA polymerase β-subunit gene (rpoB) genes to confirm the diagnosis of the isolates. The results revealed that all the tested isolates were positive for both genes. In conclusion, the incorporation of both phenotypic and genotypic characterization of C.pseudotuberculosis is more valuable and accurate for identification of isolates. Moreover, PCR is a fast and specific diagnostic tool used for the genetic analysis of C. pseudotuberculosis.