Outbreaks of highly pathogenic avian influenza (HPAI) among poultry as well as wild birds are of continuing major public concern, not only because of high economical losses but also due to lethal infections in humans. Control of the infection relies on rapid detection and identification of the causative virus strain which is carried out currently primarily by real-time RT-PCR and DNA sequencing. In a pandemic, however, the analysis of very large numbers of samples may become necessary within a short period. A method is described for the characterisation of avian influenza virus (AIV) subtypes by restriction fragment mass fingerprint (RFMF) analysis. Amplified genomic fragments encoding the pathogenicity-determining region of the hemagglutinin gene were digested with a cocktail of restriction enzymes, and the restriction fragments were assayed by mass spectrometry. Characteristic spectra with sequence coverage ranging from 75 to 100% were obtained for a panel of 27 isolates representing 18 relevant serotypes. Three marker masses were identified that are highly specific for strains of the H5N1 virus. Within the H5N1 serotype, discrimination of individual strains was possible by detailed evaluation of the spectra. The procedure described is rapid, inexpensive and compatible with automation.