The surveillance and monitoring of rotavirus (RV)-related diseases, preferably through the establishment of sentinel surveillance sites, are essential for assessing the need for vaccination and the projected results of the vaccine in terms of reducing the burden of disease. The objective of the present study was to compare RV strains isolated in Northern (Ferrara) and Southern (Galatina-LE) Italy. During 2007-2008, 115 RV-positive stool samples were collected from children with diarrhea admitted to the hospitals of Ferrara and Galatina. The specimens were genotyped for VP7 (G-type) and VP4 (P-type) gene by reverse transcription (RT) and multiplex polymerase chain reaction (PCR). A subset of 21 RV strains was randomly selected and characterized by sequence analysis of the VP7 genes. In total, seven G/P combinations (G1P[8], G2P[4], G4P[8], G9P[8], G2P[8], G1P[9], and G2P[10]) were identified. Phylogenetic comparison of the VP7 encoding gene of selected strains showed that there was similarity among RV strains circulating in Northern and Southern Italy. The observation of nucleotide sequence diversity contributes to a better understanding of RV spreading and helps to characterize the various antigenic shifts that could have an impact on vaccine effectiveness.