A study was conducted to investigate the seroprevalence and associated risk factors of Rift Valley fever (RVF) infection in cattle and some selected wildlife species at selected interface areas at the periphery of the Great Limpopo Transfrontier Conservation Area in Zimbabwe. Three study sites were selected based on the type of livestock–wildlife interface: porous livestock–wildlife interface (unrestricted); non-porous livestock–wildlife interface (restricted by fencing) and livestock–wildlife non-interface (totally absent contact or control). Sera were collected from cattle aged = 2 years representing both female and intact male. Sera were also collected from selected wild ungulates from Mabalauta (porous interface) and Chipinda Pools (non-interface) areas of the Gonarezhou National Park. Sera were tested for antibodies to Rift Valley fever virus (RVFV) using a competitive enzyme-linked immunosorbent assay (ELISA) test. AX2 test was used to assess differences between categories, and p < 0.05 was considered as significant. In cattle, the overall seroprevalence was 1.7% (17/1011) (95% confidence interval [CI]: 1.01–2.7). The porous interface recorded a seroprevalence of 2.3% (95% CI: 1.2–4.3), the non-porous interface recorded a prevalence of 1.8% (95% CI: 0.7–4.3) and the non-interface area recorded a seroprevalence of 0.4% (955 CI: 0.02–2.5), but the difference in seroprevalence according to site was not significant (p > 0.05). All impala and kudu samples tested negative. The overall seroprevalence in buffaloes was 11.7% (95% CI: 6.6–19.5), and there was no significant (p = 0.38) difference between the sites (Mabalauta, 4.4% [95% CI: 0.2–24] vs. Chipinda, 13.6% [95% CI: 7.6–23]). The overall seroprevalence in buffaloes (11.7%, 13/111) was significantly (p < 0.0001) higher than in cattle (1.7%, 17/1011). The results established the presence of RVFV in cattle and selected wildlife and that sylvatic infections may be present in buffalo populations. Further studies are