The fiber shaft of human adenoviruses (HAdVs) is essential for bringing the penton base into proximity to the secondary cellular receptor. Fiber shaft sequences of all 53 HAdV types were studied. Phylogeny of the fiber shaft revealed clustering corresponding to the HAdV species concept. An intraspecies recombination hot spot was found at the shaft/knob boundary, a highly conserved sequence stretch. For example, HAdV-D20 clustered with HAdV-D23 in the fiber shaft, but with HAdV-D47 in the fiber knob. Although all shafts exhibited the typical pseudorepeats, amino acid sequence identity was found to be as high as 92 % (interspecies) and 54 % (intraspecies). In contrast to a previous study, a flexibility motif (KXGGLXFD/N) was found in eight HAdV-D types, whereas the putative heparan sulfate-binding site (KKTK) was only found in species HAdV-C. Our results suggest that pseudotyping of gene-therapy vectors at the shaft/knob boundary is feasible, but that flexibility data of shafts should be considered.