Morchella is a nutritious and artificially cultivable rare ascomycete, and its growth and development regulation mechanisms are a current research hotspot. High-temperature stress severely limits the annual yield of Morchella, and this challenge is intensifying with global warming. However, previous studies have lacked systematic screening for heat-tolerant Morchella strains, and their molecular response mechanisms to heat stress remain unclear. In this study, we conducted a comprehensive analysis of phenotypic characteristics, physiological metabolism, and transcriptomics on 19 Morchella strains under normal (25 °C) and high-temperature (30 °C) conditions. The heat-tolerant strain HLM exhibited superior performance in mycelial growth, morphology, and field cultivation. It maintained cell homeostasis under heat stress through mild osmotic regulation (elevated levels of proline, soluble sugars, and proteins), a robust antioxidant system (increased activities of CAT, POD, and SOD), and reduced malondialdehyde accumulation. Transcriptomic analysis identified a novel regulatory model of “stress perception—metabolic preparation—terminal detoxification” in the heat-tolerant strain HLM under heat stress. The rapid upregulation of the SMPD1 gene may mediate ceramide signal generation, promoting G6PDH expression to drive carbon flow into the pentose phosphate pathway, thereby increasing NADPH output. As the detoxification terminal, AKR4C uses this reducing power to eliminate toxic carbonyl end products like malondialdehyde, completing the defense loop. These findings offer new insights into the heat-tolerance mechanisms of large ascomycetes, provide a theoretical foundation for stress-resistant Morchella breeding and cultivation in high-temperature areas, and serve as valuable resources for exploring heat-tolerance mechanisms and molecular breeding in other edible fungi.