Heterotrophic growth of microalgae could be an option to shorten their growing time and allow faster harvesting of their biomass. Suitable strains of freshwater microalgae have potential for offshore cultivation. Algae strains were isolated from rice paddies in Taiwan for heterotrophic culture. In this study, ten single strains were separated for phylogenetic and molecular evolutionary analyses. The 18S rRNA and RubisCO gene sequences were aligned and compared with the GenBank database of the National Center for Biotechnology Information (NCBI). Ten strains in five genera were identified as Leptolyngbya, Scenedesmus, Dictyosphaerium, Chlorella, and Micractinium. Under heterotrophic growth conditions, an isolated strain of Scenedesmus deserticola ICL grew faster in a high glucose concentration. The maximum concentration of S. deserticola ICLwas 3,030 mg/L and the crude lipid content was 20.5% (dry base) when the additional glucose was 20 g/L in a basic BG-11 medium. In a dark environment, the microalgae were grown using organic carbon with respiration. Organic carbon increased the specific growth rate of the microalgae efficiently.