Abstract: To explore the effects of environmental ecological factors on biodiversity of endophytic fungi, the tobacco growing areas of Enshi prefecture, Hubei province with different organic matter contents and pH values were selected. In this experiment, 6 fields with organic matter contents of 16.3, 20.7, 23.3, 27.1, 31.4, and 35.6 g/kg, respectively, and 4 fields with pH values of 4.3, 5.5, 6.4, and 7.6 respectively, were chosen. The endophytic fungi, which colonized in the inner root of tobacco, were isolated and identified with morphological characteristics and rDNA-ITS sequences. Shannon index (H') and Relative frequency (RF) were used to reflect biodiversity and distribution of the tobacco endophytic fungi. The results indicated that biodiversity of endophytic fungi increased firstly and then decreased with the increase of soil organic matter contents, and the Shannon diversity index were 0.53, 0.67, 0.82, 1.09, 0.86, and 0.59, respectively under 6 organic matter contents levels. While biodiversity of endophytic fungi decreased with the increase of pH values, and the Shannon diversity index were 1.36, 1.12, 0.93, and 0.83, respectively. We concluded that the increase of soil organic matter contents or the decrease of pH values can increase the richness of tobacco endophytic fungi in a certain range.