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Diacylglycerol (DAG) production by microbial fermentation has broad development prospects. In the present study, five endophytic fungi which could accumulate DAG were screened from <i>Taxus chinensis var. mairei</i> by using potato dextrose agar plate and flask cultivation in potato dextrose broth culture medium. The strains were biologically identified based on morphological features and semi-quantitative PCR. The identification results indicated that the five strains belonged to different genera: <i>Fusarium annulatum</i> (<i>F. annulatum,</i> coded as MLP41), <i>Trichoderma dorotheae</i> (<i>T. dorotheae,</i> coded as MLG23), <i>Colletotrichum aeschynomenes</i> (<i>C. aeschynomenes,</i> coded as MLY23), <i>Pestalotiopsis scoparia</i> (<i>P. scoparia,</i> coded as MLY31W), and <i>Penicillium cataractarum</i> (<i>P. cataractarum,</i> coded as MLGP11). The crude lipids from the strains and their corresponding triacylglycerol, 1,2-DAG, and 1,3-DAG fractions separated via thin-layer chromatography were mainly composed of palmitic acid, stearic acid, oleic acid, and linoleic acid, which in total accounted for higher than 94% of the content. The effects of fermentation conditions on the DAG productivity were discussed, and the yields of DAG were determined based on the ¹H NMR spectra of crude lipids. The highest total DAG yields of <i>F. annulatum</i>, <i>T. dorotheae</i>, <i>C. aeschynomenes</i>, <i>P. scoparia</i>, and <i>P. cataractarum</i> were 112.28, 126.42, 189.87, 105.61, and 135.56 mg/L, respectively. <i>C. aeschynomenes</i> had the strongest potential to produce DAG. The results showed that this may be a new promising route for the production of DAG via fermentation by specific endophytic fungi, such as <i>C. aeschynomenes</i>.