Abstract: Nannochloropsis are model species for investigating biofuel production by algae. Many marine Nannochloropsis genomes have been sequenced recently and their transformation systems have been established. N. limnetica is the only freshwater species, and its genome and transformation system are not yet available. In this study, a genetic transformation method for N. limnetica is established and a zeocin-resistance gene is introduced into the genome to obtain mutant library, then high oil-yield mutants are screened from the library. A vector pPha-T1-TUB that has a sh ble gene derived by Nannochloropsis β-tublin promoter and Phaeodactylum tricornutum fcpA terminator was constructed, and the linearized plasmid was introduced to N. limnetica by electroporation. Transformants were able to grow on 1 μg/mL zeocin. PCR detection indicated that 100% of the selected colonies were positive transformants and all resistant colonies contained sh ble gene after half a year. Several mutants with higher triacylglycerols content and biomass were obtained. Among them, K26 and G5 showed lower polyunsaturated fatty acid contents than wild type, which meets the requirements of the European Standard EN 14214 (2004) for biodiesel production. This study provides an efficient method to obtain advantageous mutants via generating a library of random resistant gene insertion mutations in N. limnetica.