The present study aimed to develop thermo-tolerant mutants of Chlorella pyrenoidosa NCIM 2738 for high lipids production. For this, ethyl methane sulfonate was used, which generated two effective thermo-tolerant mutants, M18 and M24 of Chlorella pyrenoidosa NCIM 2738, capable of surviving at temperature up to 47 degrees C and showing improved lipid and biomass yields. They showed 59.62% and 50.75% increase, respectively in lipid content compared to wild type at 30 degrees C, which could not grow at temperature above 35 degrees C. The novelty of this study lied in incorporation of PAM Flurometry with mutagenesis to generate thermo-tolerant mutants of C. pyrenoidosa and investigating the reasons for increased yields of mutants at cellular and photosynthetic levels with the aim to use them for commercial biodiesel production. (C) 2016 Elsevier Ltd. All rights reserved.