Two separate bird trials were performed to establish a reliable route of infection for Gallibacterium anatis in chickens, comparing intranasal (i.n.) and intravenous (i.v.) applications. Additionally, three mutually divergent isolates from three geographical locations, as shown by MALDI-TOF-MS and partial rpoB gene sequence analysis, were compared. In the first trial, birds were infected with one of the selected isolates by the i.v. or i.n. route. Subsequently, birds were killed 3, 12 and 24 h post infection following i.v. infection while at 3, 7 and 10 days post infection (dpi) in the case of i.n. infection along with birds of the control group. As a result, i.n. infection showed prominent and consistent bacterial tissue distribution in different organs persisting until 10 dpi, which was a striking contrast to the i.v. infection route. Likewise, histopathology revealed mild to severe tracheal lesions following i.n. infection. The second trial was set up to confirm both the achieved results and the robustness of i.n. infection but with an extended observation period, until 28 dpi In agreement with the preceding trial, identical results for bacteriological and histopathological examinations were obtained with persistency of bacteria until 28 dpi Comparing the three different isolates from Mexico, China and Austria, the Mexican isolate showed a somewhat higher pathogenicity than the other strains. Consequently, pathogenesis of G. anatis strains was studied in chickens elucidating i.n. infection as the most reliable route characterized by a long-lasting bacteraemia, targeting the respiratory and reproductive tract.