Herpes simplex virus type 2 (HSV-2) infection is one of the most common sexually transmitted infections (STIs) worldwide. While glycoprotein G-2 enzyme-linked immunosorbent assays are commonly used for the serological detection of HSV-2 antibodies, they have low specificity in developing countries. The Euroline Western blot (WB) is a commercially available assay that is easy to perform; however, little is known about its performance characteristics. This study evaluated Euroline WB for the detection of HSV-2 antibodies compared with University of Washington Western blot in three geographically different regions: Baltimore, MD, USA; Rakai, Uganda; and Kunming, China. Among the 135 American men attending a STI clinic in Baltimore, MD, 72% (n = 97) were HSV-2-positive by Euroline WB, showing a sensitivity of 97.8% and a specificity of 81.8%. Among the 273 commercial sex workers in Kunming, 62.3% were HSV-2-positive by Euroline WB (sensitivity 96.9%, specificity 89.1%). Among the 437 Ugandans in Rakai, 67.3% were HSV-2-positive by Euroline WB (sensitivity 98.7%, specificity 65.4%). The Euroline WB has a consistently high sensitivity, but specificity varied significantly among the different locations.