Recent and important studies have pointed out that environmental stress can represent a factor of strong risk for amphibian populations in Europe and, in particular, for the Italian complex of water frogs Rana (Pelophylax) lessonae. The analysis of vocalization in a single species can be an useful cognitive tool to better understand his behaviour and an interesting start point for conservation strategies. In the last twenty years, an increasing amount of attention has been dedicated to study of vocalization of European water frogs populations (Rana ridibunda, R. lessonae and their viable, fertile hybrid R. esculenta), in order to provide additional tool of biogeographic survey, and to underline the similarities and differences among several populations. Only one study has attempted to discuss the taxonomic question of the species that living in Central Italy using a bioacoustic approach, in synergy with other techniques. In our work, we want to provide a methodological contribution for the bioacoustic characterization of the green frog populations, through a sampling protocol and a connected technical analysis. Two green frog populations were investigated and more than 3200 calls in the field, during three breeding seasons, are recorded. The acoustic records have been performed in two sites with different environmental features and with a comparable number of male specimens. The results of this analysis has allowed to highlight the inter and intra-population differences. Furthermore, we provide a description of the individual sequences of vocalizations, spectral and temporal features of mating and territorial calls.