Two real-time PCR assays for detection of Mycolasma hyopneumoniae (Mhyop) in clinical lung samples were established and validated in parallel. One is targeting a repetitive DNA element (REP assay) the other a putative ABC transporter gene (ABC assay). The two assays were shown to be 100% specific when testing pig lungs from defined negative farms. When investigating defined positive farms the REP assay tested with a sensitivity of about 50%, the ABC assay with 90%. The two assays together, however detected 100% of positive farms. Within a single positive farm on average 90% of the samples tested positive with the REP or ABC assay. Analysing a set of 41 lungs from infected pigs from routine diagnostic the REP assay detected 50% and the ABC assay 70%, while both assays together had a sensitivity of 85%.