Abstract The diversity of environmental mycobacteria was studied in water-logged acidic forest soil. Mycobacteria were assessed in upper and lower soil horizons and summer and winter seasons using T-RFLP and sequencing of 16S rRNA gene produced with Mycobacterium-specific primers. Mycobacteria diversity differed between both the two seasons and soil horizons. Cloning revealed the presence of mycobacteria belonging to three major clusters recognized within the genus, i.e. fast-growing, intermediate, and slow-growing species, with unprecedented abundance and diversity of the latter. Two novel clusters of sequences unrelated to the known mycobacteria were identified. This study raises the possibility that forest wetlands could serve as environmental reservoirs for an unexplored diversity of mycobacteria including those related to pathogenic species.