The potential association between xenotropic murine leukemia virus-related virus (XMRV) and prostate cancer and chronic fatigue syndrome (CFS) has been much debated. To help resolve the potential role of XMRV in human disease, it is critical to develop sensitive and accurate reverse transcriptase (RT)-PCR assays to screen for the virus. Single-round RT-PCR assays were developed on the automated m2000™ system for detection of the pol or env regions of XMRV in whole blood, plasma, urine cell pellets and urogenital swab samples. Assay performance was assessed by testing two blinded panels, one comprised of whole blood and the other of plasma spiked with serial dilutions of XMRV-infected tissue culture cells and supernatant, respectively, prepared by the Blood XMRV Scientific Research Working Group (SRWG). For both whole blood and plasma panel testing, the assays showed excellent specificity and sensitivity as compared to the other tests included in the SRWG phase I study. Analytical specificity of the assays was also evaluated. Neither pol nor env PCR assays detected a panel of potential cross-reactive microorganisms, although some cross-reaction was observed with mouse genomic DNA. Screening of 196 normal human blood donor plasma, 214 HIV-1 seropositive plasma, 20 formalin-fixed paraffin-embedded (FFPE) prostate cancer specimens, 4 FFPE benign prostate specimens, 400 urine pellets from prostate cancer patients, 166 urine pellets from non-prostate cancer patients, and 135 cervical swab specimens, detected no samples as unequivocally XMRV positive.