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Abstract Background ω-3 polyunsaturated fatty acids (PUFAs) are synthesized from α-Linolenic acid (ALA, C18:3ω3) and play important roles in anti-inflammatory and antioxidant responses in mammal cells. ALA is an essential fatty acid which cannot be produced within the human body and must be acquired through diet. The purpose of this study was to evaluate the potential of a novel microalgal strain (HDMA-20) as a source of ω-3 PUFAs including ALA and eicosatetraenoic acid (ETA, C20:4ω3). Method Phylogenetic Neighbor-Joining analysis based on 18S ribosomal DNA sequence was used to identify the microalga strain HDMA-20. Autotrophic condition was chosen to cultivate HDMA-20 to reduce the cultivation cost. GC-MS was used to determine the fatty acid composition of HDMA-20 lipid. Results A microalgal strain (HDMA-20) from Lake Chengfeng (Daqing, Heilongjiang province, China) was found to accumulate high content of ω-3 PUFAs (63.4% of total lipid), with ALA and eicosatetraenoic acid (ETA, C20:4ω3) accounting for 35.4 and 9.6% of total lipid, respectively. Phylogenetic analysis based on 18S ribosomal DNA sequences suggested that the HDMA-20 belonged to genus Monoraphidium (Selenastraceae, Sphaeropleales) and its 18S rDNA sequence information turned out to be new molecular record of Monoraphidium species. The biomass productivity and lipid content of HDMA-20 were also investigated under autotrophic condition. The biomass productivity of HDMA-20 reached 36.3 mg L− 1 day− 1, and the lipid contents was 22.6% of dry weight. Conclusion HDMA-20 not only represent an additional source of ALA, but also a totally new source of ETA. The high content of ω-3 PUFAs, especially ALA, of HDMA-20, makes it suitable as a source of nutrition supplements for human health. In addition, HDMA-20 exhibited good properties in growth and lipid accumulation, implying its potential for cost-effective ω-3 PUFAs production in future.