A study was conducted in two primary health facilities in Kigali, Rwanda, to determine whether dried blood spots (DBS) used for quality control of HIV testing would give comparable results with serum after being stored for a period of 14 days and 30 days at ambient temperature. DBS and serum specimens were collected from patients undergoing HIV testing. ELISA performed on serum at baseline (gold standard) was compared with DBS results. The study included a total of 491 patients, comprising 92 (19%) males and 399 (81%) females with a median age of 27 years. A total of 148 individuals (30%) were HIV-positive. The average ambient temperature under which DBS specimens were stored at the health facilities was 23 degrees C (range 18-25 degrees C). The kappa statistic at 14 days and 30 days was 0.99 (99.4% agreement) and 0.98 (99.2% agreement), respectively, signifying almost 'perfect agreement (P<0.001)' with the gold standard. In a resource-limited sub-Saharan African country embarking on scaling-up of HIV testing, DBS stored at ambient conditions for up to 1 month were found to be a useful and robust tool to perform quality control of rapid HIV testing at the health centre level.