Polymerase chain reaction (PCR) is routinely used to confirm the presence of Mycobacterium avium subsp. paratuberculosis (MAP) in the diagnosis of ovine Johne's disease (OJD). In a recent study to overcome specificity issues with the current PCR we also developed an internal amplification control (IAC) to monitor the integrity of individual reactions. This is important given the opportunity to introduce PCR inhibitory substances within samples. Unlike other internal controls that require cloning and other molecular manipulations, this IAC only requires PCR capability for its production. The IAC was constructed in a two step PCR process that amplified a region of the Mycobacterium avium subsp. avium (MAA) genome that is not present in the MAP genome using composite primers made of an MAA region and an MAP region. The IAC was then incorporated in to a multiplex PCR that included a new MAP specific target to increase specificity. The analytical sensitivity of the IAC and multiplex PCR was established prior to evaluation on DNA samples that had been previously examined for OJD. The IAC had no adverse effects on the analytical sensitivity of the MAP specific multiplex PCR. The new PCR test was successfully used to determine the presence/absence of MAP in 25 faecal samples with known OJD status and simultaneously determine the integrity of each reaction. We present a new multiplex PCR for MAP that incorporates an IAC. The procedure used to produce the IAC is simple and highly adaptable to other PCR-based diagnostic tests.