Published by Oxford University Press on behalf of FEMS 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US. Peatlands store an immense pool of soil carbon vulnerable to microbial oxidation due to drought and intentional draining. We used amplicon sequencing and quantitative PCR to (i) examine how fungi are influenced by depth in the peat profile, water table and plant functional group at the onset of a multiyear mesocosm experiment, and (ii) test if fungi are correlated with abiotic variables of peat and pore water. We hypothesized that each factor influenced fungi, but that depth would have the strongest effect early in the experiment. We found that (i) communities were strongly depth stratified; fungi were four times more abundant in the upper (10-20 cm) than the lower (30-40 cm) depth, and dominance shifted from ericoid mycorrhizal fungi to saprotrophs and endophytes with increasing depth; (ii) the influence of plant functional group was depth dependent, with Ericaceae structuring the community in the upper peat only; (iii) water table had minor influences; and (iv) communities strongly covaried with abiotic variables, including indices of peat and pore water carbon quality. Our results highlight the importance of vertical stratification to peatland fungi, and the depth dependency of plant functional group effects, which must be considered when elucidating the role of fungi in peatland carbon dynamics.