Human adenoviruses (HAdVs) are common pathogens associated with a variety of clinical manifestations. Although most infections are self-limiting, HAdVs can cause severe or lethal infections in immunocompromised as well as in healthy individuals. Several HAdVs have recently been characterized as emerging pathogens. In Italy, epidemiological, and especially molecular epidemiological, information on this pathogen is scarce. This study describes the characterization by cell culture, PCR and phylogenetic analysis of HAdV strains originating from a small collection of clinical samples gathered between 2008 and 2009. The distribution of different HAdV species was studied and the possible presence of newly emerging types was ascertained. A broad-range primer pair was used, targeting a portion of the hexon gene, in combination with species-specific primer pairs targeting a portion of the fiber gene. Human and animal reference AdV strains were included in the study. The broad-range assay identified all HAdV strains (study and reference samples), as well as three out of four animal AdV reference strains. Seven different types belonging to three HAdV species (B, C and F) were identified in the study samples. Species C was by far the most frequent. Two co-infections were detected, each with two serotypes within species C (types 1/2 and 2/6). The combined use of these two PCR assays--allowing not only the identification of known types but also, potentially, the discovery of newly emerging ones--can provide valuable epidemiological information on the spread of HAdVs.