Molecular methodologies were used to investigate free-living fungal communities associated with biological soil crusts (BSCs), along km-scale transects on the Colorado Plateau (USA). Two cyanobacteria-dominated crust types that did not contain significant lichen cover were examined. Fungal community diversity and composition were assessed with PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting and sequencing, and fungi-specific quantitative PCR was used to measure fungal population densities as compared with those of bacteria. Our results clearly indicate that free-living fungi, while ubiquitous in BSCs, are less diverse and contribute far less biomass than their bacterial counterparts. Biological soil crust fungal community structure differed from that of uncrusted soils in their surroundings. Phylogenetic analyses placed the majority of BSC fungi within the Ascomycota, confirmed the importance of dematiaceous fungi, and pointed to members of the genera Alternaria and Acremonium as the most common free-living fungi in these crusts. Phylotypes potentially representing novel taxa were recovered, as were several belonging to the Basidiomycota that would not have been readily recognized by culture-dependant means.