Adaptive laboratory evolution is a powerful tool for microorganism improvement likely to produce enhanced microalgae better tailored to their industrial uses. In this work, 12 wild-type strains of Tisochrysis lutea were co-cultivated under increasing thermal stress for 6 months. Indeed, temperature was oscillating daily between a high and a low temperature, with increasing amplitude along the experiment. The goal was to enhance the polyunsaturated fatty acid content of the polar lipids. Samples were taken throughout the evolution experiment and cultivated in standardized conditions to analyze the evolution of the lipid profile. Genomic analysis of the final population shows that two strains survived. The lipid content doubled, impacting all lipid classes. The fatty acid analyses show a decrease in SFAs correlated with an increase in monounsaturated fatty acids (MUFAs), while changes in polyunsaturated fatty acid (PUFAs) vary between both photobioreactors. Hence, the proportion of C18-MUFAs (18:1 n-9) and most C18-PUFAs (18:2 n-6, 18:3 n-3, and 18:4 n-3) increased, suggesting their potential role in adjusting membrane fluidity to temperature shifts. Of particular interest, DHA in polar lipids tripled in the final population while the growth rate was not affected