Phospholipase D (PLD) is the major virulence factor in Corynebacteruim pseudotuberculosis that exhibit synergistic haemolysis (SH) of sheep blood cells in the presence of products from Rhodococcus equi. To evaluate the correlation between SH activity and the actual concentration of PLD involved in culture supernatants of C. pseudotuberculosis obtained from sheep with Caseous Lymphadenitis (CLA) and buffaloes with Oedematous Skin Disease (OSD). Fourteen isolates of C. pseudotuberculosis isolated from sheep with CLA and buffaloes with OSD "biotype 2" were identified by standard microbiological techniques and by multiplex PCR assay for direct detection of 16S rRNA gene, rpoB gene and pld gene specific for identification of C. pseudotuberculosis isolates. SH titers of all isolates were assayed by plate technique. The presences of PLD gene in supernatants of all isolates were performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblot technique. The concentration of PLD gene was assayed by scanning the bound PLD gene with specific antibodies that appeared at 31.5 kDa. Results presented that multiplex PCR is rapid and specific for detection of C. pseudotuberculosis isolates and there is no correlation between titer of SH activity and the actual PLD genes concentration in culture supernatants. Moreover, the SH activity of PLD genes produced by biotype 2 was generally higher than those by biotype 1.