Rotavirus is a major cause of severe acute gastroenteritis in infants and young children worldwide. It is responsible for 40% of childhood hospitalization. It has been estimated that about 23% of diarrhoeal death occurs due to the rotavirus infection in India. To study the epidemiology of rotavirus infection in children less than 5 years of age at a tertiary care teaching hospital. A total of 240 stool samples were collected from children <5 years of age suffering from acute diarrhea at a tertiary health care referral hospital in Western Uttar Pradesh, India. Rotavirus antigen was detected in stool by Enzyme immunoassay (EIA) which utilizes monoclonal antibodies directed against VP6 antigen. Molecular genotyping was done by nested multiplex PCR. The rotavirus antigen positivity rate was found to be 14.58% in this study. There was male preponderance and the male: female ratio was 1.5:1. Rotavirus diarrhoea was reported predominantly (42.85 %) in the age group of 13 to 24 months. The most common circulating G/P genotype strain was G9P[4] accounting for 36% of cases. Rotavirus remains a major cause of diarrhoea in children <5 years of age in Uttar Pradesh. However, the positivity rate has decreased after introduction of rotavirus vaccine into the Universal immunization program (UIP) in UP.