The chilling tolerance of commercial Lycopersicon esculentum cultivars (H2653, H722), Solanum lycopersicoides, an F1 hybrid of S. lycopersicoides × Sub-Arctic Maxi, and 25 BC2F2 lines of L. hirsutum × H722 (backcrossed twice to H722) was evaluated using a chlorophyll fluorescence assay. The ratio of the initial to the peak fluorescence (Fo: Fp) measured from fully expanded leaves was chosen as an indicator of plant health. Chilling induced an increase in Fo: Fp that was correlated with the sensitivity of the plant to low-temperature stress. Values of Fo: Fp remained low for cold-treated S. lycopersicoides and the F1 hybrid, which showed few symptoms of chilling-related damage, whereas the commercial cultivars, which were essentially intolerant to low temperatures, had large increases in Fo: Fp. A full range of Fo: Fp values was measured in the 25 BC2F2 lines, indicating that some chilling tolerance from the L. hirsutum parent was expressed by plants in these populations.