Coal mine drainage (CMD) collected from Meitanba mine (Hunan province, China) was studied. Coal, sulfur, ferrous sulfate, and a mixture of sulfur and ferrous sulfate were employed as selective energy substrates to culture CMD cells, and the bacterial biodiversities of each group were investigated. Four groups, C group [coal (15%, m/v) + CMD], S group [sulfur (10%, m/v) + CMD], SF group [sulfur (5%, m/v) + ferrous sulfate (22.35%, m/v) + CMD], and F group [ferrous sulfate (44.7%, m/v) + CMD] were set up. A total of 211 clones were recovered and evaluated by amplified ribosomal DNA restriction analysis (ARDRA). A total of 26 different ARDRA patterns were obtained and studied as operational taxonomic units (OTUs). The results showed that the F group could not be studied because of its extremely low cell concentration. The S, SF, and C groups all obtain their OTUs of 7, 8, and 11, respectively. Although they had the same original CMD, obvious differences were detected among these three groups. Additionally, Acidithiobacillus spp. represented 79.07% of the bacterial population in the C group. Pseudomonas spp. (11.63%) and Legionella spp. (9.3%) were also detected in the C group, which was different from the S group [Acidithiobacillus spp. ( 74.99%), Pseudomonas spp. (21.67%), and Legionella spp. (3.33%)]. The compositions of the microbial community structure of the SF group [Acidithiobacillus spp. (98.42%), Pseudomonas spp. (1.54%), Legionella spp. (0%)]was also different from the other two groups, especially the percentage of Acidithiobacillus spp. and Legionella spp. Principal component analysis (PCA) revealed that the distribution of the microbial communities of the C group was more similar to the S group than the SF group. The results showed that iron could actually play an important role in the microbial community structure of CMD, especially for the selection of some important species for coal biodesulfurization.