Since temperature effects on cucumber mosaic virus (CMV) have not been extensively studied, we examined the effects of a moderate increase in temperature on the virulence and the genetic diversity of CMV in Nicotiana tabacum (cv. Samsun NN). Two passage lines were initiated by inoculation of tobacco plants with a chlorosis strain of CMV (designated FFM-CMV) derived from full-length infectious CMV clones. Symptom changes in the tobacco plants were monitored during five successive passages at 25 and 33°C. At the fifth passage, tobacco plants inoculated with FFM-CMV at 33°C showed typical chlorosis symptoms at 7 days post-inoculation (dpi), and the plants further developed mosaic symptoms on the upper leaves at 20 dpi. In contrast, tobacco plants inoculated with FFM-CMV at 25°C did not show changes in symptoms on the upper leaves within 20 days. Sequence analysis of the coat protein (CP) genes of FFM-CMV revealed that a variety of spontaneous sequence changes were generated from the 14th and the 18th leaves of 33°C lineage tobacco plants, but not from those of 25°C lineage tobacco plants. The major CP mutation from the upper leaves of 33°C lineage tobacco plants was an L129P mutation, indicating a change in symptoms correlating to the population composition of the mutant viruses. Although relatively low mutation rates were observed from 33°C-lineage tobacco plants, some CP mutations may have been selected for systemic movement of CMV at the higher temperature, providing evidence for the importance of temperature in the process of genetic diversification of CMV populations.