Mycelium of Phytophthora infestans, the causal agent of potato late blight, can initiate crop infections over successive years by overwintering in infected potato tubers that survive as seed in fields or within cull piles. This study used four different genotypes of P. infestans to evaluate the influence of freezing temperatures on survival of mycelium in vitro. Sporangium-free mycelium of P. infestans US1, US8, US11, and US14 growing on rye agar plates was exposed to temperatures ranging from -20 and 0°C (experiment A) for different periods up to 24 h and from -5 and 0°C (experiment B) for periods up to 5 days. Cultures were incubated at 12°C after exposure, and survival of the cultures was estimated after 28 days by a digital image analysis technique that measured the average reflectance intensity (ARI) of images of the mycelium of temperature-treated cultures. The ARI values of treated cultures were compared with the growth of mycelium in negative controls (mycelium not present) and positive controls (mycelium exposed to 12°C for an equivalent period), and determination of recovery was based on statistical differences from the controls. There were significant differences in ARI values among genotypes, temperature treatment, and exposure periods in all experiments. An index of recovery was calculated for each genotype at all treatment temperatures and exposure periods for both experiments. In experiment A, exposure of mycelium of P. infestans (all genotypes) to -20 and -10°C proved lethal for exposure periods of more than 1 h. All genotypes showed some degree of recovery up to 24-h exposure at -5 and -3°C. In both experiments, exposure of mycelium of P. infestans to 0°C was not lethal to any genotype tested for any exposure period. In experiment B, all of the genotypes survived exposure up to 3 days at -3°C to some degree, but at -5°C, exposure of 1 day was lethal to all genotypes. Tolerance of freezing temperatures by mycelium of P. infestans may be an ecologically important survival mechanism and the increased tolerance of US8 and US14 may explain their predominance in cooler climates such as north-central United States.