A new tobamo-like virus was isolated from a greenhouse-grown cucumber that showed severe mosaic distortion on leaves and fruit, in the southern part of Japan. The virus was tentatively designated Cucumber mottle virus (CuMoV) and further characterized. The size and antigenicity of the coat protein (CP) and the complete sequence of the genome were compared with those of the known cucurbit-infecting tobamoviruses: the W and SH strains of Cucumber green mottle mosaic virus (CGMMV), the C and Y strains of Kyuri green mottle mosaic virus (KGMMV), Cucumber fruit mottle mosaic virus (CFMMV), and Zucchini green mottle mosaic virus (ZGMMV). The CP of CuMoV migrated more slowly than those of CGMMV-W and -SH and KGMMV-C and -Y in sodium dodecyl sulfate polyacrylamide gel electrophoresis. In Western blot analysis, the CP of CuMoV cross-reacted weakly with antisera against CGMMV-W and did not react with antisera against KGMMV-Y. The overall nucleotide sequence of CuMoV had 62.5 to 63.5% identity with those of CGMMV-W, -SH, KGMMV-Y, CFMMV, and ZGMMV. The genome organization was characteristic of tobamoviruses, encoding a 131-kb protein, a 188-kb protein, a movement protein (MP), and CP in 5′ to 3′ order. In the phylogenetic analyses of the CP, CuMoV was placed in a separate lineage from CGMMV-W, -SH, KGMMV-C, -Y, CFMMV, and ZGMMV. The results indicate that CuMoV is a distinct tobamovirus species which represents a third sub-subgroup in the cucurbit-infecting tobamoviruses.