Mycoplasma hyopneumoniae (Mhp) is the etiologic agent of mycoplasma pneumonia in swine. The purpose of this study was to develop a species-specific polymerase chain reaction (PCR)-based diagnostic reagent for the identification of Mhp. Based upon DNA sequence analysis of a cloned fragment of Mhp DNA, PCR primers were constructed and tested against different strains of Mhp, Mycoplasma flocculare, other mycoplasma species, and non-Mollicute micro-organisms which commonly inhabit the respiratory tracts of swine. A total of 40 field isolates from Mhp and four field isolates of M. flocculare have been examined. Positive signals were obtained in PCR with Mhp reference strains and all 40 Mhp field isolates, but not with other Mollicutes micro-organisms.