A cross sectional study was conducted from November 2016 to May 2017 in Bambasi and Assosa districts in Benshangul Guzum Regional State with the objectives of isolation, identification and determination antimicrobial susceptibility profile of Pasteurella multocida isolated from cattle infected with Hemorrhagic Septicemia. The study was also aimed to detect the agent using classical Polymerase chain reaction. Specimens were collected purposively from cattle in areas with outbreak of Hemorrhagic septicemia and a total of 384 swab samples were collected aseptically from cattle and transported to Assosa Regional Veterinary Laboratory using a cold chain system. Specimens were cultured on MacConkey agar and blood agar. Different biochemical tests were also used in order to identify and characterize the agent of the suspected disease. The prevalence of P. multocida was 13 (3.39%) out of 384 samples tested). All the isolates were subjected to the in vitro antimicrobial ssensitivity tests and the result indicated the presence of different degrees of susceptibility and resistance to most of the antibacterial agents. The degree of susceptibility ranges from 15.4% for Tetracycline upto 61.5% for Sterptomycin, and resistance is between 15.4% for Sterptomycin, Clindamycin and Chloramphicol and 69.2% for Tetracycline. Similarly, (60 %) of this discs showed the presence of multidrug resistance. The result of this test clearly indicates that, Sterptomycin, Clindamycin and Chloramphicol could be prospective drugs of choice. Analysis of PCR assay revealed the presence of P. multocida serotype B2. In conclusion, the presence of P. multocida serotype B2 in the selected areas can be considered as potential pathogens in causing Hemorrhagic septicemia in cattle especially where there is no any practice of vaccination. Therefore, strict measures like proper vaccination and antibiogram test to select effective drugs should be regularly implemented.