Equine arteritis virus (EAV) establishes persistent infection in the stallion reproductive tract, and the carrier stallion continues to shed virus in semen for weeks to years or lifelong. The objective of this study was to elucidate the intra-host evolution of EAV during persistent infection in stallions. Seven EAV seronegative stallions were experimentally infected with EAV KY84 strain and followed for 726 days post-infection, and sequential clinical samples including semen were collected for virus isolation and next-generation sequencing (NGS). In addition, archived sequential semen samples from two stallions that were naturally infected with EAV KY84 for a long-period (up to 10 years) were also sequenced by NGS. The data demonstrated genetic bottleneck event and selection during acute infection followed by intra-host quasispecies diversification during persistent infection in the stallion reproductive tract. Also, the full-length genome of a novel EAV donkey strain from Chile and a noncytopathic bovine viral diarrhea virus-1 (ncpBVDV-1) strain contaminating rabbit kidney-13 cells were also sequenced by NGS. The EAV donkey strain was genetically distinct but antigenically cross-reacted with EAV antisera, and it was phylogenetically closely related to the South African donkey strain of EAV. Genetic and phylogenetic analyses demonstrated that ncpBVDV-1 belongs to BVDV-1b group.