River biofilms are natural communities composed of both autotrophic and heterotrophic microorganisms which play an important role in nutrient and energetic cycles in aquatic systems. As other living organisms, biofilms are likely to be impacted by diffuse environmental pollution. Rivers in urban areas have indeed been shown to be contaminated by mixtures of chemicals from urban and industrial wasterwaters or rainwaters. It is generally not easy to estimate the biological effects of such chronic but non-lethal contamination. Yet, biofilms, which respond rapidly to environmental stress, could be used as early-warning indicators of pollution. Diffuse urban pollution might result in modifications of biofilm diversity, of bacterial and eukaryotic community structures, in the loss of pollutant-sensitive microbial species or in the appearance of resistance genes and possibly modifications of bacterial and eukaryotic activities (photosynthesis, biodegradation processes, etc.), thus resulting in a disruption of the ecological balance of aquatic ecosystems. A recent study on freshwater biofilms has shown that diffuse urban exposure could lead to tolerance acquisition using the Pollution-Induced Community Tolerance (PICT) approach. Indeed non-lethal exposure to a toxic agent leads to an increase in community tolerance, either by adaptation of the species or by selection of resistant species. Metal tolerance of heterotrophic communities was measured using a short-term toxicity test based on β-glucosidase activity. An increase in metal tolerance (Cd, Cu, Ni, Pb and Zn) was observed for natural biofilms collected in the Seine river upstream to downstream from the urban area of Paris (north of France). The aim of this study was to investigate more closely the molecular mechanisms involved in metal tolerance acquisition at the community level. Indeed, bacteria have developed different resistance mechanisms to survive under metallic stress. The presence of metal resistance genes within microbial communities of natural river biofilms was investigated by PCR amplification. Primers targeted genes encoding efflux pumps involved in trace metal resistance (czcA: resistance to Co, Zn and Cd, cadA: Cd, copA: Cu and silA: Ag) or in Hg speciation changes (merA). First, three different DNA extraction methods were evaluated by comparing the yield and purity of DNA extracted from two biofilm samples collected at two different sampling sites in the Seine river basin (one site is located in a small third-order river and the other in a large six-order river). An extraction method combining bead-beating and phenol/chloroform extraction was chosen (the other methods were a commercial kit designed for samples with high humic acid content and a protocol involving chemical lysis and chloroform/isoamyl alcohol extraction). PCR protocols were optimized on DNA extracted from both samples and revealed the presence of metal resistance genes in both communities. Second, we investigated the presence of metal resistance genes in biofilms collected at three sites located along the Seine river (upstream and downstream from the area of Paris) at different seasons (autumn, spring, summer). The presence of metal-resistance genes was interpreted in connection with tolerance levels measured on the same biofilm samples and with the metallic contamination gradient caused by the urban area of Paris.