Abstract: Isochrysis galbana serves as a critical live feed in aquaculture hatcheries. Summer high temperatures often trigger algal decline, leading to feed shortages and increased production risks. A high-temperature-tolerant mutant strain (MT) of I. galbana, screened by our research group, exhibits superior growth performance, with significantly higher cell density and total protein content at high temperatures compared to the wild-type strain (WT). To elucidate its thermotolerance mechanism, this study integrated physiological and biochemical indicators with lipidomics to systematically compare growth, physiology, and lipid metabolism between MT and WT. The results showed that during days 1-5, the specific growth rate of MT at 30 ℃ (0.51±0.03) d⁻¹ was significantly higher by 55% and 76% ompared to WT at 30 ℃(0.33±0.05) d⁻¹ and 20 ℃(0.29±0.06) d⁻¹, respectively (P＜0.05). Meanwhile, at 30 ℃, MT maintained the highest levels of pigments and protein content: chlorophyll a (0.30±0.02) pg/cell, chlorophyll c (0.090±0.008) pg/cell, carotenoids (0.058±0.005) pg/cell, and protein (4.26±0.06) pg/cell, which were 1.22-fold and 1.63-fold higher than those of WT at 20 ℃ and 30 ℃. This suggests that MT may sustain photosynthetic efficiency and cell proliferation under high temperature by enhancing the synthesis of photosynthetic pigments and proteins. Lipidomic analysis revealed that MT at 30 ℃ significantly accumulated monogalactosyldiacylglycerol (MGDG), a key lipid for the photosynthetic membrane system, thereby helping maintain membrane stability under high temperature. Storage triacylglycerol (TG) was also accumulated in large amounts, providing energy reserves for sustained growth. This lipid remodeling, characterized by coordinated enhancement of membrane stability and energy storage, may represent a molecular strategy of MT for adaptation to high-temperature environments. This study preliminarily reveals the physiological and lipid metabolic responses of the high-temperature I. galbana mutant, providing theoretical support for research on heat-stress adaptation mechanisms in microalgae and for the development of stress-resistant feed microalgae.