Although a number of protocols have been developed for detection of viruses at the genus or family level, universal approaches to detect and identify unknown viruses are still required. High-resolution tandem mass spectrometry was used to identify accurately peptide masses and their constituent sequences from partially purified plant virus preparations. Analysis of the peptide fragment masses against a virus database using pattern-matching algorithms identified sequences with homology to known virus peptides and also predicted peptides using de novo sequence analysis. This method provided sufficient information to confirm the identity of two known viruses that were included as controls (Cucumber mosaic virus and Tomato spotted wilt virus) and to identify unknown viruses in six viral isolates. The unknown viruses have been identified as four common viruses (Alfalfa mosaic virus, Tobacco streak virus, Citrus leaf blotch virus and Ribgrass mosaic virus), and two novel viruses (a potexvirus and a vitivirus). The identification of viruses from five distinct families by the tandem mass spectrometric determination of their coat protein demonstrates that this is a useful method for initial virus identification. This method, complemented with molecular or immunological procedures, provides a rapid and convenient way to identify both known and novel plant viruses.