Regional and global studies of ectomycorrhizal fungal (EMF) communities associated with Alnus have made progress in determining the key factors that influence EMF diversity and biogeography. Smaller scale studies provide a valuable complement by relating EMF to environmental gradients and describing how community composition is influenced by competition and niche partitioning. We examined controls on EMF community composition in an 80 y-old Alnus rubra stand. EMF species were identified using root tip morphology and DNA sequencing, and related to soil variables (soil moisture, total C, total N, C:N ratio, PO4-P, soil pH) and to Frankia nodulation using Threshold Indicator Taxa ANalysis (TITAN) and multivariate techniques. Twenty-two EMF species were identified, including 14 that are new associates of A. rubra. EMF community composition varied temporally and was influenced by Frankia nodulation and soil chemistry. Species co-occurrence patterns suggest niche partitioning and competitive exclusion interact with subtle differences in the soil environment to influence EMF community composition.