The surveillance network RotaNet-Italia was established in 2007 in order to investigate the diversity of co-circulating rotavirus strains in Italy, and to provide a baseline for future assessment of possible effects of vaccine implementation in selecting novel versus common rotavirus strains. A total of 2,645 rotavirus strains from pediatric patients with acute diarrhea were collected over three consecutive seasons from September 2006 through August 2009, and partially characterized by standardized multiplex RT-PCR. Most of strains (89.1%) belonged to genotypes G1-G4, and G9, associated with either P[8] or P[4], commonly found in humans worldwide. However, in at least 2.0% of cases, viruses exhibited either a G or P type typical of animal viral strains, suggesting gene reassortment events between rotaviruses of different origin. Mixed infections with two or more rotavirus strains were observed frequently (7.6% of patients), and depended on the frequencies of co-circulating rotaviruses of one particular genotype. The numbers and genotypes of likely natural reassortants of common genotype rotaviruses were found to be correlated with the observed numbers and genotypes of mixed infections. Large variation in the relative frequency of different rotavirus genotypes was observed between different seasons and/or areas of Italy, suggesting independent evolution or differential introduction of viral strains with respect to both time and space.