Rotavirus is the most common cause of severe diarrhea in children worldwide. Monitoring the diversity of rotavirus strains is of great importance for current and future vaccination programs. To determine the diversity of rotavirus circulating in Asuncion, Paraguay, between 2006 and 2007, we carried out a molecular characterization of rotaviruses detected in children <5 years old and adults (>18 years old). We found that the most common circulating strain was G2P[4] (69/143), followed by G9P[8] (37/143). The temporal distribution of strains showed that, in children, G2P[4] was predominant in 2006, and that G2P[4] and G9P[8] were co-predominant in 2007, whereas in adults, G2P[4] was predominant in both years. Additionally, one G9P[6] and three G12P[9] strains were found in adult samples, making this the first report of these strains circulating in Paraguay. Sequence analysis of the G12P[9] strains suggests across-border migration of this strain within the southern cone of America.