Disclosed herein is a PCR-based assay for Mycoplasma hyopneumoniae, a species-specific primer pair for use in the assay, and a related diagnostic kit. The primer pair is made up of an oligonucleotide having the nucleotide sequence 5'-AAGTTCATTCGCGCTAGCCC-3' and an oligonucleotide having the nucleotide sequence 5'-GCTCCTACTCCATATTGCCC-3'. Preferably, the kit contains an oligonucleotide probe having the sequence 5'-GGTAGCCCTTCCTTTGAGGT-3'.