BACKGROUND: Viral suppression is a key indicator of antiretroviral therapy (ART) response among HIV-infected patients. Dried blood spots (DBS) are an appealing alternative to conventional plasma-based virologic testing, improving access to monitoring in resource-limited settings. However, validity of DBS obtained from fingerstick in field settings remains unknown. OBJECTIVES: Investigate feasibility and accuracy of DBS vs plasma collected by healthcare workers in real-world settings of remote hospitals in Malawi. Compare venous DBS to fingerstick DBS for identifying treatment failure. STUDY DESIGN: We recruited patients from ART clinics at two district hospitals in Malawi, collecting plasma, venous DBS (vDBS), and fingerstick DBS (fsDBS) cards for the first 149 patients, and vDBS and fsDBS only for the subsequent 398 patients. Specimens were tested using Abbott RealTime HIV-1 Assay (lower detection limit 40 copies/ml (plasma) and 550 copies/ml (DBS)). RESULTS: 21/149 (14.1%) had detectable viremia (>1.6 log copies/ml), 13 of which were detectable for plasma, vDBS, and fsDBS. Linear regression demonstrated high correlation for plasma vs. DBS (vDBS: ß=1.19, R(2)=0.93 (p<0.0001); fsDBS ß=1.20, R(2)=0.90 (p<0.0001)) and vDBS vs. fsDBS (ß=0.88, R(2)=0.73, (p<0.0001)). Mean difference between plasma and vDBS was 1.1 log copies/ml [SD: 0.27] and plasma and fsDBS 1.1 log copies/ml [SD: 0.31]. At 5000 copies/ml, sensitivity was 100%, and specificity was 98.6% and 97.8% for vDBS and fsDBS, respectively, compared to plasma. CONCLUSIONS: DBS from venipuncture and fingerstick perform well at the failure threshold of 5000 copies/ml. Fingerstick specimen source may improve access to virologic treatment monitoring in resource-limited settings given task-shifting in high-volume, low-resource facilities.