A total of 402 fecal specimens from infants and children with acute gastroenteritis in five places (Tokyo, Maizuru, Saga, Sapporo, and Osaka) in Japan from July 2003 to June 2004 were collected and then tested for the presence of rotavirus by RT-PCR. Of these, 83 were positive for rotavirus and this accounted for 20.6%. Rotavirus was further characterized to G-types (VP7 genotypes) and P-types (VP4 genotypes). Interestingly, an emergence of rotavirus G3 was identified with an exceptionally high prevalence (97.5%; 81 of 83), followed by rotavirus G2 (2.5%; 2 of 83). The P-types of 19 rotavirus strains, which could not be typed by RT-PCR, were determined as P[8] with multiple point mutations at the VP4 primer-binding site by sequencing analysis. The predominant genotype was G3P[8] (95.2%, 79 of 83), followed by a number of unusual combinations G3P[4] (2.4%, 2 of 83), and G2P[8] (2.4%, 2 of 83). Another interesting feature of the study was the demonstration of a great genetic diversity in new variant rotavirus G3 strains circulating in Japan. In comparison with rotavirus G3 strains circulating in 1990-1995 in Japan, a wide range of amino acid substitutions (up to 16) of new variant rotavirus G3 VP7 genes was identified. Of note, the changes at positions 96, 99, and 100 were revealed to be located in the antigenic region A, and 213 in the antigenic region C. To the best of our knowledge, this is the first reporting of an emergence of new variant rotavirus G3 together with a sudden disappearance of G1, G4, and G9 in infants and children with rotavirus infection-associated gastroenteritis in Japan.