Mosquito-borne viruses are an important cause of human and domestic diseases worldwide. As crucially important emerging pathogens, they have caused multiple, notable and unnoticed epidemics of human disease over recent decades. All mosquito-borne viruses circulate within sylvatic cycles among wildlife and forest mosquitoes and may cause diseases in rural and urban populations after spillover transmission to human and domestic animals. The aim of this study was to determine mosquito diversity and transmission of Rift Valley fever virus (RVFV), dengue virus (DENV) and Chikungunya virus (CHIKV) in South-eastern Tanzania. In the present cross-sectional study, a total of 1320 adult mosquitoes were collected using Biogent (BG) sentinel traps and battery-powered aspirators. In addition, a total of 31 larvae were collected using a dipping technique and allowed to hatch into adults. Morphological identification of mosquitoes showed the presence of 12 different species which were unevenly distributed among different ecological zones within the two districts. Culex mosquitoes were found to be dominant at all mosquito collection sites and represented 89.1% followed by 10.1% Aedes and 0.8% Mansonia. Aedes mosquitoes were pooled into groups of 20 mosquitoes resulting into a total of 12 pools. No mosquito pool was positive for DENV or CHIKV when Aedes mosquito pools were screened for these viruses. However, RVFV was found in two pools of mosquitoes including one of Aedes aegypti and another of Aedes pembaensis. Furthermore, when Aedes mosquito pools were screened, eight pools were positive for Flavivirus and six pools were positive for Alphavirus respectively. The positivity to Flavivirus and Alphavirus with concurrent absence of DENV and CHIKV in these mosquitoes indicates the presence of viruses other than DENV and CHIKV. Results obtained from this study show that mosquitoes in Kilombero Valley carry viruses of iii serious public health importance. It is recommended that mosquito-borne viruses be screened in febrile patients presenting at points of care in order to improve prognosis.Southern African Centre for Infectious Disease Surveillance (SACIDS)