Respiratory tract infections in sheep are among the important health problems that affect all sheep ages around the world. Nine bacterial isolates obtained from sheep with respiratory tract infections were selected to be used in the current study. The isolates included 3 Staphylococcus aureus, 4 Klebsiella pneumoniae, and 2 Pseudomonas aeruginosa. Following the primers design by the Primer3Plus software tool and optimization of the conventional polymerase chain reaction (PCR), the primers were validated for their use in the multiplex PCR experiments. The MFEprimer program was used to check the suitability of the primer set combinations for multiplex PCR. The MFEprimer software was successful in designing the multiplex-PCR experiments and determining the optimal primer set combinations. Multiplex PCR was able to amplify specific DNA sequences of one, two or three target genes of these mixed microorganisms in the same PCR reaction tube. This technique efficiently detected combinations of two organisms, either S. aureus with K. pneumoniae, S. aureus with P. aeruginosa or K. pneumoniae with P. aeruginosa. Moreover, multiplex PCR was also able to detect the presence of the three organisms together in the same reaction tube. To conclude, this study confirmed multiplex-PCR as a specific, sensi- tive, rapid, accurate, and cost-effective molecular diagnostic method for identification and differentiation of three clinically important bacteria associated with sheep respiratory tract infections, including S. aureus, P. aeruginosa, and K. pneumoniae. This can efficiently support control and treatment of such diseases and would increase the economy of the animals’ owners and wellbeing of the animals.