Although tolerance to high temperature is crucial to the summer survival of Iris germanica cultivars in subtropical areas, few physiological studies have been conducted on this topic previously. To remedy this, this study explored the physiological response and expression of heat shock factor in four I. germanica cultivars with varying levels of thermotolerance. The plants’ respective degrees of high-temperature tolerance were evaluated by measuring the ratio and area of withered leaves under stress. Several physiological responses to high temperatures were investigated, including effects on chlorophyll, antioxidant enzymes, proline, and soluble protein content in the leaves of four cultivars. CaCl2 was sprayed on ‘Gold Boy’ and ‘Royal Crusades’ considered being sensitive to high temperatures to study if Ca2+ could improve the tolerance, and LaCl3 was sprayed on ‘Music Box’ and ‘Galamadrid’ with better high-temperature tolerance to test if calcium ion blocker could decrease their tolerance. Heat shock factor genes were partially cloned according to the conserved region sequence, and expression changes to high-temperature stress with CaCl2 or LaCl3 treatments were thoroughly analyzed. Results showed that high temperature is the primary reason for large areas of leaf withering. The ratio and area of withered leaves on ‘Music Box’ and ‘Galamadrid’ were smaller than ‘Gold Boy’ and ‘Royal Crusades’. CaCl2 slowed the degradation of chlorophyll content and increased proline and soluble protein in ‘Gold Boy’ and ‘Royal Crusades’ but had no significant effect on activating peroxidase or superoxide to improve high-temperature tolerance. Genetic expression of heat shock factor in ‘Gold Boy’ and ‘Royal Crusades’ was upregulated by Ca2+ at later stages of leaf damage under high-temperature stress. LaCl3 down-regulated the physiological parameters and expression level of heat shock factor in ‘Music Box’ and ‘Galamadrid’. These results suggest that different I. germanica cultivars have varying high-temperature tolerance and furthermore that Ca2+ regulates their physiological indicators and expression level of heat shock factor under stress.