Algal biofuel feedstocks are excellent candidates for sustainable and eco-friendly fuels for the next generation, which can be improved by genetic modifications for their maximal production of biomaterials. However, currently available genetic modifications involve the introduction of foreign DNA into the algal genome, and this may face legal and public conflicts due to the risk of environmental, economic, and/or health problems. In this regard, we employed an old concept of crop improvement that has been accepted in the long history of agriculture, i.e. polyploidization. Polyploidization of crop plants has been selected fortuitously or intentionally not only for increased quality and/or quantity of products, but also for enhanced stress tolerance. We induced diploidy in the model algae Chlamydomonas by treating haploid cells with the microtubule inhibitor colcemid, and the resulting diploids were selected for increased colony size and neutral lipid contents. Two of the isolated diploid strains containing doubled DNA contents, named CMD ex1 and CMD ex4, were increased in their cell size and cellular weight. These diploids were excellent in coping with abiotic stresses, including nutritional, oxidative, and cold stresses. Under these conditions, the diploids accumulated two times more biomass and FAME yield compared to the control. To understand underlying mechanisms, we performed RNA-Seq analyses for the diploid under the cold stress. Transcriptomic analyses revealed that the diploids showed enhanced expression of genes involved in photosynthesis, energy metabolism, and translation as well as reduced starch metabolism. Overall, diploids of Chlamydomonas showed improvements including increased yields of biomass and FAME and enhanced stress tolerance compared to wild-type organisms. The results demonstrate that polyploidization can be utilized in industrial microalgae for the production of biofuels and other biomaterials not only on a laboratory scale but also in outdoor cultivation, where stress conditions are inevitable.