In this study, the effect of heat stress pretreatment factors on cell suspension culture of potato was investigated. The duration of stress pretreatment at 5–50 °C for 20 min was evaluated by measuring cell growth and viability using the trypan blue exclusion method. Data were collected by measuring the dry weight of suspended cells, duration of heat stress, growth, and biomass content at the respective time points. Cell viability was negatively affected by 85 to 25% after pretreatment with heat stress between 35 and 50 °C. However, suspended cells exposed to 30 °C and 35 °C during the recovery period began to regain viability at 75% and 69%, respectively, after 55 h. In suspended cells treated at 40 °C or high temperatures, a complete decrease in cell viability was observed, resulting in no recovery of growth. In potato cell suspension cultures, heat treatments at 40 °C or above for 20 min not only have an immediate effect on cell viability, but also lead to subsequent cell death. Effective viability tests showed the natural heat tolerance of potato plants in cell suspension cultures. The basic parameters of cellular response to heat determined in this study will be helpful in selecting heat-tolerant potato varieties by establishing in vitro cell suspension cultures.