Summary A multiplex polymerase chain reaction (PCR) was used to detect mycoplasma infection in human DNA samples of patients with CFS and related illnesses. One set of oligonucleotide primers which are specific for a highly conserved region among all members of the genus Mycoplasma along with three other primer sets which are specific for Mycoplasma fermentans, M. hominis, and M. penetrans species were used in this assay. The sensitivity of detection was determined by adding known mycoplasma DNA copy numbers to 1 μg of genomic DNA from healthy subjects. Each sample was subjected to 40 cycles of amplification. The detection level was determined to be 7, 7, 9, and 15 mycoplasma DNA copies per μg of human genomic DNA for M. genus, M. fermentans, M. hominis, and M. penetrans, respectively. The assay was applied to DNA extracted from the PBMCs of individuals suffering from chronic fatigue syndrome (CFS) (n = 100), fibromyalgia (FMS) (n = 40), rheumatoid arthritis (RA) (n = 60), and gulf war syndrome (GWS) (n = 60) and compared to age- and sex-matched healthy individuals (n = 160). The percentage of M. genus infection detected in CFS, FMS, RA, and GWS was 52,54, 49, and 55%, respectively. M. fermentans was detected in 32, 35, 23, and 36%, M. hominis was detected in 9, 8,11, and 5%, and M. penetrans was detected in 6, 4, 7, and 3% of CFS, FMS, RA, and GWS patients, respectively. M. genus, M. fermentans, M. hominis, and M. penetrans were detected in 15, 8, 3, and 2% of healthy matched controls. This assay provides a rapid and cost efficient procedure to screen clinical samples for the presence of three potentially pathogenic species of Mycoplasma with a high level of sensitivity and specificity.