Understanding the variability of acoustic signals is a first important step for the comprehension of the evolutionary processes that led to current diversity. Herein, we evaluate the variability of the advertisement call of the phyllomedusid species from the genera Phyllomedusa Wagler, 1830 and Pithecopus Cope, 1866 at different levels: intra-individual, intra-population, inter-population, intra-species, and inter-specific. An analysis of coefficients of variation showed a continuum of variability between the acoustic parameters analyzed, from static to highly dynamic. The majority of the variation was attributed to the inter-specific level, while call parameters at the intra-individual level varied the least. However, each parameter behaved differently with call interval being the most variable across all levels. Most temporal acoustic parameters were affected by environmental temperature, while pulse rate and dominant frequency were strongly influenced by body size. Only pulse rate was correlated to the geographic distance between populations, while all parameters presented a significant phylogenetic signal. Based on these results, we discuss the possible importance of different evolutionary forces and the usage of vocalizations for taxonomic purposes.