West Nile Virus (WNV)-specific nucleic acid amplification testing (NAAT) of organ and tissue donors remains controversial. We report three years of WNV donor screening in Alberta Canada using NAAT. Between 2003 and 2005, 1549 initial specimens were received. A valid negative result was issued within the specified turnaround time on 1531 (98.8%). The initial NAAT was successful for 1393 samples (90%), while repeat testing using an alternate NAAT resolved a further 126 samples. For 12 of 14 donors, a second specimen provided a valid negative result. Failure to generate a valid negative result in time resulted in rescheduling of one living related organ transplant, and surgery proceeded in the absence of a final result in one multi-organ donation after risk assessment. For 11 tissue donors, tissues were discarded due to lack of a WNV result. Invalid results usually occurred on postmortem haemolyzed tissue donor samples due to inhibitory reactions. There were no confirmed positive donors, no false-positive results and no solid organs lost due to WNV testing. We conclude that WNV NAAT of organ and tissue donors can be implemented without compromising availability of donors but requires committed laboratory support.