Neonatal diarrhea is the main cause of morbidity and mortality in calves, and Rotavirus is the main viral etiology. The objective of the current study was to study the epidemiological role of Rotavirus infection in diarrheic neonatal cattle and buffaloes 'calves in Assiut Governorate, Egypt. From December 2015 to November 2019, a total number of 315 neonatal calves belonged to different localities of Assiut Governorate, Upper Egypt, were clinically examined. Fecal samples of investigated calves were subsequently collected and serologically tested to reveal-up Rotavirus infection by using latex agglutination test (LAT) and immunochrmatographic assay (ICA). The seropositive samples by LAT and ICA were 16.74% and 8.54%, respectively. The clinical findings of Rotavirus infection in enteric calves were pointed. The percentage of Rotavirus infection was 14.92% (44/295) of clinically diarrheic calves. However, 10% (2/20) of apparently healthy calves (n = 20) harbor Rotavirus in their feces suggesting carrier status. The high percentage of Rotavirus infection (20.13%) was obviously observed in 3 days-4 weeks old calves. There were no significance differences (P < 0.05) between the percentages of Rotavirus infection and sex, species (cattle and buffaloes) and breed (Native and Cross breed) of serologically tested calves. Moreover, there is no significant variations (p < 0.05) between calves under farmer’s hand and calves bred in farm in susceptibility to Rotavirus infection. Based on climatologic conditions of Assiut, seropositive cases were more prevalent (P < 0.001) in cold months (22.67%) than the warm and hot months (7.27%) in Assiut.