We determined the effects of atmospheric temperature (10–30 ± 2°C in 5°C increments) and carbon dioxide (CO2) levels (400 ± 50 ppm, 540 ± 50 ppm, and 940 ± 50 ppm) on the infection of Solanum tuberosum cv. Chubaek by Potato leafroll virus (PLRV). Below CO2 levels of 400 ± 50 ppm, the PLRV infection rate and RNA content in plant tissues increased as the temperature increased to 20 ± 2°C, but declined at higher temperatures. At high CO2 levels (940 ± 50 ppm), more plants were infected by PLRV at 30 ± 2°C than at 20 or 25 ± 2°C, whereas PLRV RNA content was unchanged in the 20–30 ± 2°C temperature range. The effects of atmospheric CO2 concentration on the acquisition of PLRV by Myzus persicae and accumulation of PLRV RNA in plant tissues were investigated using a growth chamber at 20 ± 2°C. The M. persicae PLRV RNA content slightly increased at elevated CO2 levels (940 ± 50 ppm), but this increase was not statistically significant. Transmission rates of PLRV by Physalis floridana increased as CO2 concentration increased. More PLRV RNA accumulated in potato plants maintained at 540 or 940 ± 50 ppm CO2, than in plants maintained at 400 ± 50 ppm. This is the first evidence of greater PLRV RNA accumulation and larger numbers of S. tuberosum plants infected by PLRV under conditions of combined high CO2 levels (940 ± 50 ppm) and high temperature (30 ± 2°C).