As in most of the African continent, the status of hepatitis E virus (HEV) infection in domestic animals in São Tomé and Príncipe, an archipelago off the western equatorial coast of Central Africa, is also completely unknown. In the present study, we investigated the presence of HEV among domestic animals in São Tomé and Príncipe. A total of 93 stool samples from different animal species (goat, cow, pig, chicken, duck, and monkey) were tested for HEV RNA using two real-time RT-PCR assays, followed by a nested RT-PCR assay for sequencing and phylogenetic analysis. A total of six samples (1 cow stool and 5 pig stools) were found to be positive for HEV RNA of which one pig stool was positive by broad spectrum nested RT-PCR. Phylogenetic analysis showed that the retrieved sequence clustered within HEV subgenotype 3f, similar to zoonotic strains of European countries and posing interesting questions on past introduction of European HEV into São Tomé and Príncipe archipelago. This is the first report describing the presence and molecular characterization of HEV in São Tomé and Príncipe.