Territorial calls (hooting) of two tawny owl subspecies, Strix aluco aluco (the eastern European subspecies) and Strix aluco willkonskii (the Caucasian subspecies), were recorded from October 2007 to February 2013, to study variation between individuals and subspecies. Calls of the eastern European subspecies were recorded in Moscow region (Russia) and “Gomolshanskie Lesa” National Park (Ukraine). Males of the Caucasian subspecies were recorded in “Bolshoi Utrish” Reserve and “Sochinskii” National Park (Russia). The territorial calls of the eastern European and Caucasian subspecies are compared for the first time. The discriminatory accuracy of individual calls reached 98% for S. a. aluco and 77% for S. a. willkonskii when accounting for six frequency and temporal parameters of their call. We found the call of the two subspecies to differ in half of the acoustic parameters considered, suggesting that macrogeographic variation between the Eastern European and Caucasian subspecies may have taken place.