Invasive alien species (IAS) potentially may alter all levels of the ecological organization of aquatic water bodies. Therefore, in the context of the EU Water Framework Directive, they represent a significant pressure that should be considered in the assessment of the ecological status of a water body and in the formation of restorative programs. A study was carried out to examine different sampling techniques and to assess the current population structure and the differences in spatial distribution of the alien crayfish Orconectes limosus at four sites in Lake Varese (Northern Italy). Three methods were used to assess the crayfish population: mark-recapture, catch per unit effort during night-time snorkelling and quadrat sampling. For the mark-recapture exercise the ventral somites of crayfish were marked using visible implant elastomer tags. A laboratory experiment was used to verify the effectiveness of elastomer tags for the species prior to their use in the field. Crayfish were more abundant at sites characterized by stone substrate than at sites dominated by macrophytes. Hydromorphological alteration of the shoreline (quantified using the newly developed lake habitat survey methodology) at one site appeared to promote the establishment of alien crayfish. Crayfish caught by snorkeling in open water led to an underestimation of the smallest size-classes compared with underwater quadrats. Underwater quadrats on stone substrates provide a quantitative estimation of abundance and size-structure allowing the comparisons of crayfish populations from different lakes. This method could be applied to assess the extent of pressure resulting from alien crayfish under the Water Framework Directive. Crayfish biomass was found to be 82.9% of other macroinvertebrates indicating their importance in the structure and functioning of the lake ecosystem. The presence of a second alien species of crayfish, Procambarus clarkii, was recorded for the first time in the lake.