Amplified fragment length polymorphisms (AFLPs) were used to characterize the genotypic diversity of a total of 114 Gallibacterium anatis isolates originating from a reference collection representing 15 biovars from four countries and isolates obtained from tracheal and cloacal swab samples of chickens from an organic, egg-producing flock and a layer parent flock. A subset of strains was also characterized by pulsed-field gel electrophoresis and biotyping. The organic flock isolates were characterized by more than 94% genetic similarity, indicating that only a single clone was apparent in the flock. The layer parent flock isolates were grouped into two subclusters, each with similarity above 90%. One subcluster contained only tracheal isolates, while the other primarily included cloacal isolates. In conclusion, we show that AFLP analysis enables fingerprinting of G. anatis, which seems to have a clonal population structure within natural populations. There was further evidence of clonal lineages, which may have adapted to different sites within the same animal.