Five sugarcane leaf samples exhibiting mosaic symptoms and collected in 1999–2003 in Bangladesh, India, Sri Lanka, Thailand, and Vietnam tested negative in serological assays (ELISA and TBIA) using antiserum to sugarcane mosaic virus (SCMV) and a potyvirus specific monoclonal antibody. In addition, these samples reacted negative in RT-PCR assays with SCMV, sorghum mosaic virus (SrMV), and potyvirus specific primers. Mosaic symptoms were reproduced after mechanical inoculation of sugarcane, maize, and sorghum plants with diseased leaf extracts, but symptoms were less severe than those of SCMV and SrMV. Electron microscopy of partially purified virions from inoculated plants showed flexuous filaments characteristic of the Potyviridae family. The five sugarcane leaf samples tested positive in RT-PCR assays with two primer pairs designed to detect sugarcane streak mosaic virus (SCSMV), and in immunological assays with SCSMV antisera. SCSMV was detected by ELISA with three different antisera and by RT-PCR in 34 leaf samples from 30 cultivars exhibiting mosaic symptoms and originating from six Asian countries (the five mentioned above and Pakistan). The sequences of the RT-PCR amplicons (376 nucleotides) showed 98.5–100% identity with SCSMV isolates from GenBank. The immunological, molecular, and pathogenic characteristics of SCSMV were different from those of SCMV and SrMV. Our results suggested that SCSMV was already widespread in Asia more than two decades ago. Furthermore, immunological variation occurs among SCSMV isolates, which needs to be considered for serological diagnosis of the virus. RT-PCR primers developed in this study proved to be very efficient for detection of SCSMV from different countries.