Twenty-five isolates of Mortierella spcies were prepared, which can be used for the production of polyunsaturated fatty acids (PUFAs)-rich oil for nutritional supplements. The fatty acid contents were determined after heterotrophic fermentation. The content of total fatty acids (TFAs) in the cell dry weight of all isolates including two commercially purchased Mortierella alpina strains ranged from 207.51 to 370.11 mg/g, whereas PUFAs were the dominant fatty acid type. The highest PUFA-containing strain, M. alpina SC9, was identified and confirmed as a new strain of M. alpina through comparison analysis of the sequences of internal transcribed spacers 1 and 2 (ITS1 and ITS2) and the 5.8S rDNA region. During a 7-day fermentation, the PUFAs content of M. alpina SC9 varied between 189.83 and 240.00 mg/g, with a remarkable correlation between the oleic acid (C18:1, OA) and arachidonic acid (C20:4n-6, AA) contents and between the linoleic acid (C18:2n-6, LA) and AA contents, suggesting the PUFA content in the fungus is tightly regulated. This study provides a framework of isolation, identification, and characterization of an important PUFA-producing species, M. alpina.