Field recordings of the calling song and of an amplitude modulated signal produced by males of Cicada barbara from North Africa and the Iberian Peninsula were analysed in order to assess the geographical acoustic variation and the potential usefulness of acoustic data in the discrimination of subspecies and populations. Sound recordings were digitized and the frequency and temporal properties of the calls of each cicada were analysed. In all regions studied, peak frequency, quartiles 25, 50 and 75% and syllable rate showed low coefficients of variation suggesting inherent static properties. All frequency variables were correlated with the latitude, decreasing from south to north. In addition, most acoustic variables of the calling song showed significant differences between regions, and PCA and DFA analyses supported a partitioning within this species between Iberian Peninsula+Ceuta and Morocco, corroborating mtDNA data on the same species. Therefore, the subspecific division of C. barbara into C. barbara barbara from Morocco and C. barbara lusitanica from Portugal, Spain and Ceuta finds support from the present acoustic analyses, a result which is also reinforced by molecular markers.