Mycoplasma suis is a haemotrophic facultative pathogen of swine which can cause acute or chronic infections. Its accurate diagnosis is often hampered by the lack of sensitive and specific methods, therefore a real-time PCR based assay was developed using PCR amplification of a 101 bp fragment and a minor groove binder probe which could detect <2 copies of the 16S ribosomal RNA gene of M. suis. In comparison to a previously developed nested PCR technique the real-time was more specific and more sensitive. It detected several strains of M. suis but no other Mycoplasma species of pigs. Probe-based PCR was more specific than SYBR-Green using only the primers (without the probe) and detected M. suis in 76 of 372 blood samples and 18 of 224 spleen samples. Samples positive with nested PCR or SYBR-Green but negative with the probe (n=4) turned out to be false positive results. Field samples could be quantified using a standard curve of a cloned PCR amplicon. Real-time PCR can be used to quantitatively detect M. suis DNA in blood and spleen samples from infected pigs, supporting the diagnosis of eperythrozoonosis in the field.