Equine influenza (EI) is a highly contagious viral respiratory disease affecting equids, with the potential of causing widespread outbreaks across Europe, the Americas, Asia, and other regions of the world. In Tunisia, in the spring of 2021, an EI virus outbreak occurred in a farm housing purebred Arabian horses that exhibited respiratory signs. This outbreak led to a national lockdown on horse movements and the cancellation of equestrian events. A total of 432 serum samples, collected from 2018 to 2021, were tested using ELISA competition assay. In addition, 100 Nasal swabs were collected during the 2021 outbreak and tested for the presence of EI via qRT-PCR. Five of these samples underwent full sequencing targeting hemagglutinin (HA) and neuraminidase (NA) genes. Phylogenetic and phylogeographic analyses were carried out on HA and NA gene sequences using Bayesian methods. Out of 432 serum samples, 80 tested positive, leading to an overall seroprevalence of 18.51 %, with the highest values recorded in 2018 and 2021. Complete sequences of the HA and NA genes identified the subtype as H3N8, belonging to the American lineage, Florida clade 1. Tunisian strains exhibited close genetic relationships with those from Algeria, the USA, France, Saudi Arabia, and the UK. Phylogeographic analysis reveals probable origin of the virus in Tunisia from the USA and France in 2017-2018. Our study highlights the critical need for continuous surveillance and molecular characterization of EI strains, particularly given their high transmissibility and potential for global dissemination.