Mycoplasma fermentans is a likely causative agent of HIV-associated nephropathy. In a pilot study, M. fermentans DNA was detected with polymerase chain reaction (PCR) in urine samples from renal allograft recipients; nine (39.1%) out of 23 renal allograft recipients (most of whom had chronic allograft rejection) and none of the 20 controls, were infected with M. fermentans. A cross-sectional study was conducted to investigate the prevalence of M. fermentans in urine samples from renal allograft recipients. Midstream urine samples were centrifuged at 13,000 x g, purified with QIAamp and tested with PCR using RW004/RW005 and an internal control to screen for the presence of inhibitors. Of the 264 participants recruited, 263 completed the questionnaire (172 men, 92 women); 53 had chronic renal allograft rejection, 106 had chronic renal dysfunction without rejection, 69 had a normal renal allograft for more than 3 months and 35 had a renal allograft for less than 3 months. All urine samples yielded positive results for the internal control. Mycoplasma fermentans DNA was detected once i prospectively collected urine samples. The only individual infected with M. fermentans was also seropositive for HIV-1. This study demonstrates that M. fermentans can be at most sporadically detected in urine from patients living with a renal allograft but is not implicated in chronic rejection of allograft.