There is little information on the epidemiology of Human Adenoviruses (HAdVs) in Italy. In this study, 103 HAdV isolates, collected by the A. Gemelli Hospital (Catholic University Medical School of Rome, Italy) between 1987 and 2005, were genotyped by sequencing and phylogenetic analysis on a partial hexon gene region. Nine different serotypes belonging to all six HAdV species were identified. Serotype 2 was the most frequent (53.4%), followed by serotype 1 (15.53%) and serotype 41 (9.7%). Partial-hexon-based identification was confirmed as an effective tool for studying the molecular epidemiology of HAdVs.