BACKGROUND: World Health Organization guidelines now recommend routine use of viral load testing, where available, for patients receiving antiretroviral treatment (ART). However, its use has not been routinely implemented in many resource-limited settings due to cost, availability and accessibility. Viral load testing is complex, making its application in resource-limited settings challenging. We describe the issues encountered by Medecins Sans Frontieres (MSF) when using routine viral load testing in a large HIV programme in sub-Saharan Africa. METHODS: Between October 2005 and August 2006, more than 1200 patients on ART had viral load tests at baseline and at three-month intervals performed by a local reference laboratory that was quality assured by an experienced international institution. Concerns with reliability of results halted testing. The quality control measures instituted with a second laboratory and outcomes of these were documented. RESULTS: In 2005 and 2006, only 178 of 334 (53%) previously ART-naive patients tested after six to 12 months of treatment had viral loads of less than 1000 copies/mL. Similar MSF programmes elsewhere demonstrated virological suppression rates of more than 85%, and duplicate testing showed unacceptable discordance. Laboratory problems encountered included: disregarded quality control; time delays; requirement for retesting; and duplicate sample variations. Potentially harmful clinical outcomes of inaccurate viral load results include: unnecessary ART regimen changes; unnecessary enhanced adherence counselling after “false failures”; and undetected virological failure. CONCLUSIONS: Viral load testing performed without rigorous quality control carries the risk of erroneous and potentially damaging results. Viral load testing should be utilized only if robust quality assurance has been implemented. Our experience in this and other settings led to the development of a guide for assessing the suitability of a laboratory for viral load testing that can be used to help achieve reliable results.