Rift Valley fever virus (RVFV) is an emerging biodefense pathogen that poses significant threats to human and livestock health. To date, the interepidemic reservoirs of RVFV are not well defined. In a longitudinal survey of infectious diseases among African buffalo during 2000-2006, 550 buffalo were tested for antibodies against RVFV in 820 capture events in 302 georeferenced locations in Kruger National Park, South Africa. Overall, 115 buffalo (21%) were seropositive. Seroprevalence of RVFV was highest (32%) in the first study year, and decreased progressively in subsequent years, but had no detectable impact on survival. Nine (7%) of 126 resampled, initially seronegative animals seroconverted during periods outside any reported regional RVFV outbreaks. Seroconversions for RVFV were detected in significant temporal clusters during 2001-2003 and in 2004. These findings highlight the potential importance of wildlife as reservoirs for RVFV and interepidemic RVFV transmission in perpetuating regional RVFV transmission risk.