IntroductionRift Valley fever (RVF) is a mosquito-borne transboundary zoonotic disease with major implications for animal and public health. Following the 2000 outbreak in Saudi Arabia and Yemen, the risk of reintroduction through cross-border livestock movement remains a continuing concern. This study aimed to estimate the seroprevalence of Rift Valley fever virus (RVFV) among illegally introduced livestock intercepted in the Jazan region of southwestern Saudi Arabia and to assess the added value of combined serological and molecular testing for detecting recent infection under routine surveillance conditions.MethodsCross-sectional study was conducted using routine diagnostic data from confiscated livestock intercepted between January 2017 and December 2019.Results and discussionA total of 2,202 serum samples from sheep, cattle, and camels were screened using a commercial competitive ELISA kit (ID.vet, France). Seroreactive samples were further characterized using IgM ELISA and real-time polymerase chain reaction (RT-PCR). Seroprevalence estimates were calculated with 95% confidence intervals (CI) and by species and year of collection. Agreement between IgM ELISA and RT-PCR was evaluated using Cohen’s kappa coefficient. Overall, 38 of 2,202 samples were cELISA-positive, giving an RVFV seroprevalence of 1.7% (95% CI: 1.3–2.4). Of the Seroreactive samples, 6/38 (15.8%; 95% CI: 7.4–30.4) were IgM-positive, indicating recent infection. Three samples were RT-PCR-confirmed, and all molecularly confirmed cases were also IgM-positive. Agreement between IgM and PCR was moderate (κ = 0.62). Seroprevalence was higher in sheep than cattle, but without statistical significance (OR = 1.61; 95% CI: 0.82–3.17; p = 0.190). These findings provide surveillance-based evidence of RVFV exposure and recent infection among livestock intercepted at a high-risk border interface. They support illegal cross-border livestock movement as a potential pathway for RVFV introduction, while not establishing the geographic source or transmission route. Strengthening integrated laboratory surveillance, border biosecurity, and One Health coordination is essential for early detection and risk reduction.