Objective: We examined the blood of 48 North American Chronic Fatigue Syndrome (CFS) patients subsequently diagnosed with Lyme Disease Borrelia burgdorferi and compared these to 50 North American CFS patients without evidence of Borrelia burgdorferi infections for presence of Mycoplasma spp. co-infections using forensic polymerase chain reaction. Results: We found that 68.75% of CFS/Lyme patients show evidence of mycoplasma co-infections (Odds Ratio=41.8, Confidence Limits=11.26-155.16, p<0.001) compared to controls, whereas 50% of CFS patients without a diagnosis of Lyme Disease Borrelia burgdorferi show mycoplasma co-infections (OR=19.0, CL=5.25-68.78, p<0.001 compared to controls). Since CFS patients without a diagnosis of Lyme Disease have a high prevalence of one of four Mycoplasma species and a majority show evidence of multiple infections, we examined CFS/Lyme patients’ blood for various Mycoplasma species. We found that CFS patients with Lyme Disease Borrelia burgdorferi mostly had single species mycoplasma infections (OR=31.67, CL=8.63-116.16, p<0.001) with a preponderance of M. fermentans infections (50% of patients, OR=59.0, CL=7.55-460, p<0.001), whereas the most commonly found Mycoplasma spp. in CFS patients without Lyme Disease was M. penumoniae (34% of patients. OR=14.94. CL=3.25-68.73, p<0.001). Conclusions: The results indicate that a subset of CFS patients show evidence of infection with Borrelia burgdorferi, and a large fraction of these patients were also infected with Mycoplasma fermentans and to a lesser degree with other Mycoplasma species.