Most studies dealing with individual pattern in acoustic signals are based on recordings made within a few hours or a few days. However, in long-lived animals, individual signatures are likely to be involved in social contact and pairing over several years. Variability of calls and more particularly of individual specific pattern over years has been poorly investigated. This study examines calls produced by a nocturnal long-lived raptor, eagle owls (Bubo bubo) in order (1) to determine which call structures are individually specific and (2) to examine variability of these individual structures over 2 years. A computerised sound analysis and subsequent data were submitted to univariate and multivariate statistics to determine individual specific cues of the call. A first analysis conducted on 116 calls emitted by 9 wild males recorded on a short time period enables to identify each emitter without any mistake. Analysis conducted on 70 calls emitted by 5 captive birds recorded during two different years emphasised a weak variation of all call parameters. Discriminant analysis correctly classified 100% of the individual eagle owls and thus, it is possible to reliably assign a call to the individual caller based on the acoustic properties of the vocalisation. We therefore conclude that territorial calls emitted by eagle owls may constitute an individual pattern over several years. The possible biological role of individual signatures and their use for monitoring population purpose are discussed.