Corynebacterium pseudotuberculosis is a facultative intracellular bacterium that causes caseous lymphadenitis in sheep and goats. This study was designed to evaluate the validity of PCR assay protocol for the direct detection of C. pseudotuberculosis in 147 samples of lymph nodes (prescapular and mediastinal) from carcasses of naturally infected sheep and to compare its performance with the traditional bacteriological culture technique. C. pseudotuberculosis was isolated in 81 samples mainly from prescapular nodes and a specific 203 bp PCR amplified pld gene DNA fragment was detected in 85 samples. The total agreement score between the 2 methods was 95.92%, the relative PCR sensitivity and specificity to culture being 98.76% and 92.42%, respectively. The positive and negative predictive probabilities given by PCR were 98.4% and 94.1%, respectively. The C. pseudotuberculosis detection limit given by PCR was 2.1x10(4)-2.1x10(3) CFU/mL in sheep lymph tissue samples. In conclusion, the PCR assay proved to be a sensitive and rapid method for the detection of C. pseudotuberculosis in lymph node samples from naturally infected sheep.