Systemic infection of wheat plants with Soil-borne wheat mosaic virus (SBWMV) requires temperatures below 20°C. Here we examine the cause of the temperature sensitivity by inoculating infectious in vitro transcripts of SBWMV RNA1 and RNA2 to barley mesophyll protoplasts. After RNA inoculation, protoplasts were incubated at temperatures between 15 and 25°C for up to 48 h. Western blot analysis showed that the capsid protein accumulated most abundantly at 17°C but was not detectable at 25°C. Northern blot analysis showed that the wild-type RNA1 and RNA2 and their subgenomic RNAs accumulated most abundantly at 17°C but were barely detectable at 25°C. An RNA1 mutant in which the p152 and p211 replicase genes were placed between the 5′- and 3′-untranslated regions also replicated most efficiently at 17°C but not at 25°C. Thus, the requirement for temperatures lower than 20°C for SBWMV infection is primarily determined by replication of RNA1, which encodes the viral RNA replicase.