Metabarcoding is a promising DNA-based method for identifying airborne pollen from environmental samples, with potential advantages over microscopic methods. This method includes several steps and its successful outcome depends on a good DNA quality and quantity, which will be used as a starting material. Currently, there is no clear protocol for the sample preparation and the DNA extraction, especially for gravitational pollen samplers. Here, we present a methodology to analyze environmental samples collected by both volumetric (Burkard spore trap) and gravitational samplers (Tauber trap). For this, we compared different protocols (e.g. DNA extraction kits) on pure pollen (single species) and the best combination, in terms of DNA yield, was applied to environmental samples (pollen mixtures of different taxa). For the environmental samples, a short fragment (about 150 base pairs) of chloroplast DNA (cpDNA) was amplified by universal primers for plants (trnL), 30 amplicons were Sanger-sequenced and the taxonomic assignment was accomplished through a comparison to a reference custom-made database, including important widespread anemophilous taxa from the study area (Eastern Italian Alps). Having as reference the microscope results, we proved that metabarcoding is efficient in identifying taxa even at the species level, having established improved protocols and a good reference database. We plan to apply a semi-quantitative analysis by Next Generation Sequencing to assess the pollen spectra of different Natura 2000 habitats in the Alps.