Rotavirus infection is the most common cause of severe gastroenteritis during childhood worldwide, especially in developing countries. Two rotavirus vaccines are available for childhood immunization programs. Evaluation of the vaccine performance will benefit from knowledge of the epidemiological features of rotavirus infection in regional settings. Limited information on the molecular characteristics of the rotavirus types circulating in Venezuela is available. Eighty seven (89.7%) of the 97 ELISA rotavirus positive stool samples collected from children with diarrhea aged <5 years during 2003 in Valencia (Carabobo State), were G-, P- and NSP4-genotyped by RT-PCR and/or automated sequencing. Four common combinations, G3P/NSP4-E1, G2P/NSP4-E2, G9P/NSP4-E1, and G1P/NSP4-E1 were responsible for 50.6%, 35.6%, 5.7%, and 1.1%, respectively of cases of rotavirus diarrhea, most of them (66%) in children ≤12 months. One uncommon G8P/NSP4-E2 strain was also detected. Temporal fluctuation of genotype distribution occurred, but no differences by age, diarrhea severity score, sex, treatment type or patient medical attention were observed, except for the G3P/NSP4-E1, associated with a more severe dehydration than any other type (P < 0.01). The results confirm the broad diversity among rotavirus strains circulating in Venezuela prior to vaccine implementation, showing the predominance of G3, significant proportion of G2 and moderate circulation of G9 strains. Epidemiological surveillance is needed to detect the emergence of new genotypes that could escape protection induced by vaccination. J. Med. Virol. 83:2225–2232, 2011. © 2011 Wiley Periodicals, Inc.