Lettuce (Lactuca sativa L.) is grown worldwide, from temperate to subtropical climates. Spring season production in humid, subtropical regions, such as southern Florida, is characterized by rising ambient temperatures that can stress lettuce to prematurely bolt and lose shelf life. The objectives of this research were 1) to identify genetic variability in heat tolerance and shelf life among lettuce types and accessions grown under humid, subtropical conditions, and 2) to understand the genotype × environment (G × E) interaction to estimate shelf life of these lettuce accessions. Five lettuce types (romaine, crisphead, butterhead, leaf, and Latin) were grown under commercial conditions in the Everglades Agricultural Area near Belle Glade, FL, USA, for five field experiments over two seasons. Lettuce heads were evaluated at harvest, and subsets were transported to a local commercial grower/shipper for vacuum-cooling and storage at 15 °C according to previously determined protocols for accelerated shelf life testing. Visual appearance ratings were made across harvests and storage time points to segregate lettuce accessions with an estimated marketable shelf life >14 days. The breeding lines tested in this research had head weight and marketability comparable to commercial cultivars. Notably, the crisphead accessions 50113, 60157, 60159, and H1098 had the highest estimated and actual shelf life of more than 21 days, with no presence of bolting or tipburn. Meanwhile, romaine, butterhead, leaf, and Latin types had accessions with estimated and actual shelf life ranging from 14 to 28 days and no presence of bolting or tipburn. A G × E analysis indicated that this interaction is significant; therefore, breeders should consider analyzing G × E when developing new cultivars with good horticultural characteristics, longer shelf life, and most importantly, adaptation to warmer humid, subtropical conditions.