The microalgal genus of Nannochloropsis is considered one of the most promising organisms for the production of biofuels due to their high lipid content. Transformation systems for marine Nannochloropsis species have been established in the recent decade, however, genetic manipulation of Nannochloropsis limnetica, the only known freshwater species in this genus, is not yet available. Based on established marine Nannochloropsis species electrotransformation protocol, nuclear genetic transformation was established in N. limnetica, meanwhile the appropriate antibiotic selection concentration and electric field strength of electroporation were determined. For the selection of transformants in N. limnetica on plates, 0.07 mu gmL(-1) of zeocin or 5 mu gmL(-1) of hygromycin B was proved sufficient, and the transformation efficiency was <2x10(-8) with a single pulse ranging from 2200 to 2600V using 2-mm electroporation cuvettes. Pretreatment of N. limnetica with 10mM lithium acetate and 3mM dithiothreitol before electroporation increased transformation efficiency hundreds of times, and the highest transformation efficiency of 10-11x10(-6) was obtained with an electric field strength of 12,000V/cm. Our results help to expand the biotechnological applications of this freshwater species and provide means for successful electrotransformation of other microalgae as well.Graphic abstractHigh-efficiency transformation of freshwater Nannochloropsis pretreatment of N. limnetica with 10 mM lithium acetate and 3 mM dithiothreitol before electroporation increased transformation efficiency hundredsoftimes. [GRAPHICS] .