Hemorrhagic septicemia has been an acute and fatal disease among cattle and buffaloes caused by Pasteurella multocida. The prevalence of this disease is 49% in animals of Pakistan. The present study was to evaluate the prevalence, multidrug resistance and the molecular heterogeneity within clinical strains of P. multocida collected from different cities of Pakistan. Tissue samples of lung and spleen of dead animals were used for diagnostics. Rapid plate screening test for Hyaluronidase of clinical Isolates showing clear and white zones proved that given isolate is pathogenic. Amongst one hundred and ninety nine total isolates, only one hundred and one isolates were found to be virulent as clinical isolate of P. multocida. Isolates were confirmed through amplification (PCR) of specie-specific and Hemorrhagic septicemia type B causing test. The primer pair of specie -specific test showed a band of 300bp in all subspecies of P.multocida whereas the primer pair of HS type B causing test showed a band of 460bp in all HS-causing serotype of P.multocida. The high prevalence was found only in Karachi. Whole cell and envelope protein profile of the isolates on SDS-PAGE showed presence of