Microalgae-derived biomass has been considered as the most promising candidate for next generation biofuel due to its sustainability and biodegradability. In this study, microalgal strain Chlamydmonas reinhardtii was randomly mutagenized by using a chemical mutagen, ethyl methane sulfonate (EMS) to create mutants showing enhanced lipid production. We identified three random mutants that displayed high lipid production in the screening using Nile red staining. Among those, mutant #128 was selected as candidate for further studies. Our flow cytometry and confocal microscopy analysis revealed that mutant #128 contains larger and more abundant lipid bodies than that of wild-type. Moreover, mutant #128 showed 1.4-fold increased fatty acid methyl ester (FAME) content compared to wild-type under nitrogen depleted condition. In addition, mutant #128 grew faster and accumulated more biomass, resulting in high lipid production. 2D gel electrophoresis and MALDI-TOF analysis used for gene targeting revealed that beta-subunit of mitochondrial ATP Synthase and two-component response regulator PilR may be involved in enhanced characteristics of mutant #128. These results show the possibilities of EMS mediated random mutagenesis in generation of mutants to produce high amount of lipid as well as further study for molecular mechanism of mutants.