Rift Valley fever (RVF) is a mosquito-borne zoonotic disease which presents in epizootic form over large areas of a country following heavy rains and flooding, and is characterized by high rates of abortion and neonatal mortality, primarily in sheep, goats and cattle; and fatal haemorrhagic syndrome in humans. In Southern and Eastern Africa, RVF occurs at intervals of between 5-12 years and the last outbreak was in 2006/2007. Little is known on where the virus is maintained during the inter-epidemic period; therefore this study was conducted to investigate sero-prevalence of RVF in wild and domestic ruminants, and small mammals during the inter-epidemic period. Serum samples from buffaloes, gazelles, cattle and rodents were tested for specific RVF IgG antibodies using the indirect enzyme-linked immunosorbent assay (I-ELISA) based on the recombinant nucleocapsid protein (rNp) of Rift Valley Fever Virus (RVFV). The results revealed that 31.4% of buffaloes (n=102), 23.1% of Grant’s gazelle (n=13), 14.5% of Thomson’s gazelle (n=69), 19% of Impala (n=21), 20% of hartebeest (n=20), 6.5% of topi (n=31), 2.3% of wildebeest (n=88), 11.6% of cattle (n=593), 4.5% of goats (n=110) and 3.8% of sheep (n=53) were sero-positive for RVF. It was interesting to note that some sero-positive animals including 7 buffaloes, 20 cattle, 4 goats, 1 sheep, 2 topi, 2 hartebeests, 1 Grant’s gazelle and 1 impala were born 1 to 4 years after the end of the last outbreak, indicating occurrence of new infections in the absence of overt clinical signs during the inter-epidemic period. These results indicate presence and circulation of RVFV within the Serengeti ecosystem five years after the last disease outbreak. The implications of the results in relation to the role of wildlife in the ecology of RVFV, surveillance, preparedness and response to RVF are discussed.