Human infection with the xenotropic murine leukemia virus-related virus (XMRV) has been associated controversially with prostate cancer and chronic fatigue syndrome. Information is lacking about the mechanisms of transmission and potential risk groups for XMRV infection. Plasma and peripheral blood mononuclear cells (PBMCs) from individuals with retroviral infections, chronic viral hepatitis, autoimmune diseases, prostate cancer, chronic fatigue syndrome, and blood donors were tested for XMRV markers. Antibodies to XMRV proteins p15E and gp70 were examined using research assays. DNA extracted from PBMCs was tested for the presence of XMRV gag and env sequences. A total of 1103 specimens belonging to individuals with chronic fatigue syndrome and/or fibromyalgia (437), prostate cancer (69), HIV-1 (149), HTLV-1/2 (31), chronic hepatitis B (81), chronic hepatitis C (72), autoimmune diseases (18), and blood donors (246) were examined. Overall, three samples (0.3%) were p15E seroreactive (two HTLV-1 and one HCV patient). Another 15 (1.4%) were gp70 seroreactive (six chronic fatigue syndrome-fibromyalgia, four blood donors, two HIV-1, one prostate cancer, one HBV, and one HCV). Four specimens were initially positive for XMRV gag sequences, but none could be confirmed by repeated testing. In summary, no evidence of XMRV infection was found in populations with retroviral and viral hepatitis infections in Spain. Likewise, XMRV was not recognized in patients with autoimmune diseases, chronic fatigue syndrome-fibromyalgia, prostate cancer, or healthy blood donors.