Abstract: Rotavirus is mainly associated with gastroenteritis in adults and major cause of diarrhea in child’s under 5 years of age. The present study was designed to standardize the latex agglutination test with respect to agar gel precipitation test and then detection of rotavirus in sewage and drinking water by latex agglutination test. Latex agglutination test is an easy, rapid, highly sensitive and specific test for diagnosis as compared to other methods. However, for the current study n= 240 samples were collected by convenient sampling technique from different sources including half (n= 120) from sewage sources and half (n= 120) from drinking water sources. The collected samples were transferred under optimum conditions 4oC temperature in sterile falcon tubes to laboratory of Institute of Microbiology, Faculty of Veterinary Science, University of Agriculture, Faisalabad. Then samples were processed for rotavirus detection by latex agglutination test. The results were analyzed by ANOVA technique using SPSS version 20.0 of computer program. The present study showed 60% (72/120) and 26.6% (32/120) rotavirus in sewage and drinking water respectively. The present study concluded that rotavirus percentage was detected higher in sewage water then drinking water and needs precautions to avoid contamination of sewage water containing higher amount of rotavirus.