Poultry farming is crucial for the livelihoods of small-scale producers in the Northen Benin, yet the industry faces challenges from diseases like ND, which threaten both poultry health and economic stability. This study investigates the seroprevalence of Newcastle Disease Virus (NDV) among indigenous chickens and guinea fowl in the Atacora and Donga regions of Northern Benin, addressing a significant gap in regional poultry health data. A cross-sectional study was designed, sampling a total of 300 birds, including 191 indigenous chickens and 109 guinea fowl, from six districts. Systematic random sampling was employed to select smallholder farms, and blood samples were collected for Hemagglutination inhibition (HI) tests to detect NDV antibodies. Statistical analyses, including chi-square tests, determined associations and differences in seroprevalence between regions and species. Results revealed notable variations in NDV seroprevalence between the two regions. In Atacora, 59.22% of chickens tested positive for NDV antibodies, with 52.33% of non-vaccinated and 94.12% of vaccinated chickens showing positive results. In Donga, the overall seroprevalence was 46.59%, with 27.78% of non-vaccinated and 59.62% of vaccinated chickens testing positive. A chi-square test indicated a significant difference in NDV seroprevalence between the two regions for chickens (x² = 12.901; P = 0.024). For guinea fowls, seroprevalence was 63.53% in Atacora and 62.50% in Donga, with no significant difference observed (x² = 1.102; P = 0.954). This study provides the first serological data on NDV prevalence in northern Benin, highlighting the endemic nature of NDV and the critical epidemiological role of guinea fowls due to frequent exposure and field infections, especially in the Atacora region. It recommends enhancing vaccination coverage, improving biosecurity measures, and conducting further research to isolate and characterize virus strains to develop more effective control strategies.