Traditional methods used for studying communities of aquatic hyphomycetes are based on the detection and identification of their asexual spores under a microscope. These techniques limit detection to aquatic fungi present in sufficient quantity and capable of sporulating under laboratory conditions. Our objective was to develop a molecular approach to detect and monitor all types of fungi (i.e. strictly or facultatively aquatic) in harsh habitats (i.e. groundwater wells and heavily polluted surface water) where fungal biomass may become limited. We developed a semi-nested PCR protocol for fungal 18S ribosomal RNA genes coupled to subsequent analysis of the PCR products by Temperature Gradient Gel Electrophoresis (TGGE) to monitor the fungal community structure in aquatic habitats characterized by a pollution gradient. Our TGGE-protocol was compared with the traditional morphological approach and revealed a higher diversity in groundwaters and in some polluted surface waters. Thus, PCR-TGGE is a promising alternative in particular in habitats with low fungal biomass. The dynamics of fungal biomass and sporulation rates during the first weeks of leaf colonization showed that habitats with adverse ecological conditions allow only reduced fungal growth, which might subsequently impact upper trophic levels and thus interfere with key ecological processes of leaf decomposition.