Genomic subtractive hybridization of closely related Pasteurella multocida isolates has generated clones useful in distinguishing hemorrhagic septicemia-causing type B strains from other P. multocida serotypes. Oligonucleotide primers designed during the sequencing of these clones have proved valuable in the development of PCR assays for rapid species- and type-specific detection of P. multocida and of type B:2 in particular. This study demonstrated that the primer pair designed from the sequence of the clone 6b (KTT72 and KTSP61) specifically amplified a DNA fragment from types B:2, B:5, and B:2,5 P. multocida and that the primers KMT1T7 and KMT1SP6 produced an amplification product unique to all P. multocida isolates analyzed. It was also shown that PCR amplification performed directly on bacterial colonies or cultures represents an extremely rapid, sensitive method of P. multocida identification.