The increasing frequency and duration of high-temperature extremes have adversely impacted tomato production globally. An improved understanding of the physio-biochemical traits under such climatic changes will enable the selection, development, and adoption of resilient tomato varieties. Thus, this study investigated a suite of fifteen tomato plant traits that could be involved in the physio-biochemical mechanisms of thermotolerance to facilitate the selection of heat-tolerant tomato varieties. Ten tomato varieties selected from a previously published study were exposed to three temperature conditions (26/18 °C as control, 38/28 °C for 7 d as heat stress, and 40 °C for 7 h as heat-shock treatment). The results demonstrated that a composite variable of membrane injury metrics, including electrolyte leakage, malondialdehyde content, and heat injury index, was the most successful variable for discriminating varietal thermotolerance under either heat stress or heat shock conditions among all the evaluated traits. Principal components of other plant physiological traits performed poorly in distinguishing thermotolerance among the varieties and essentially did not match the results from the membrane injury composite variable. We identified ‘Celebrity,’ ‘Heat Master,’ and ‘Valley Girl’ as the most thermotolerant varieties under both heat treatment conditions, whereas ‘Arkansas Traveler’, ‘Pruden's Purple’, and ‘Tasti-Lee’ were found to be the most thermosensitive. These results echoed results from a previous open-field screening trial of 43 tomato varieties. Results from this study suggest that measurements of membrane injury are more effective than leaf physiological traits and that heat shock treatments may be as effective as longer term, moderate heat stress treatments for screening tomato thermotolerance, implying that future controlled environment screenings may reduce the number of variables measured and the length of their trials.