Internal Transcribed Spacer - Restriction Fragment Length Polymorphism (ITS-RFLP) and sequencing techniques were used to compare similarity of fungi decomposing roots of Picea sitchensis (Sitka spruce), Pinus contorta (lodgepole pine), Pinus ponderosa (ponderosa pine), Pseudotsuga menziesii (Douglas-fir), and Tsuga heterophylla (western hemlock). Fungal DNA from hyphae growing on coarse roots, cultures isolated from roots, herbarium specimens, culture collections, and field collected samples was extracted, amplified, and digested with endonucleases. Over two hundred ITS-RFLP patterns from fungal samples were separated. Linearly increasing ITS-RFLP sampling intensity curves indicate high total genetic diversity of fungi. Nei and Li similarity index analysis showed similarities of fungi on roots as measured by ITS-RFLP patterns samples ranged from 0 to 1 (i.e., 0% to 100% similar) depending upon the root species, location, and harvest stand age. Sequences were obtained for the most common ITS-RFLP patterns. Results indicate that zygomycete fungi were most prominent in decomposing woody conifer roots and that the similarity of fungi was low between tree species and locations.