Rotaviruses have been confirmed as causative agents of nosocomial gastroenteritis in children, but limited data exist concerning the epidemiology of nosocomial rotavirus gastroenteroentritis in Iran. The aim of this study was to determine the prevalence and molecular characteristics of rotavirus in children less than five years old with nosocomial diarrhea in Shahrekord (southwest of Iran).This cross-sectional study was conducted between December 2010 and October 2011. The study population consisted of children aged 6 to 60 months who were hospitalized in the pediatric ward of Hajar Hospital in Shahrekord, Iran, due to diseases other than diarrhea. Nosocomial diarrhea was defined as that occurring more than 48 hours after admission to the hospital for non-diarrheal causes. Rotavirus and G genotypes were determined by seminested reverse transcriptase polymerase chain reaction in 100 stool samples. In these 100 samples, the prevalence of rotavirus infection was 30%; the most common genotyes were G1 (20%) and G9 (20%). According to the findings of the study, genotyping of rotavirus is necessary to monitör changes in strain prevalence. Identifying strains over time could affect future vaccine strategies and detect any regional differences of genotype prevalence.