There are currently two known serotypes of equine adenovirus (EAdV), equine adenovirus type 1 (EAdV1) and equine adenovirus type 2 (EAdV2); EAdV1 is predominantly associated with upper respiratory tract infections while EAdV2 appears to have a higher association with gastrointestinal infection, however, very little is known about the prevalence of these viruses in horse populations in Australia. In this study we tested 122 serum samples obtained from horses in New South Wales, Australia, using a standard serum neutralization (SN) assay and ELISA. Ninety-seven of the 122 sera displayed had moderate to high titers of antibodies to EAdV1 and/or EAdV2. Eighteen of the 122 sera were positive for both EAdV1 and EAdV2. In contrast, only thirty-seven positive samples were detected using the ELISA. These results suggest that EAdV1 and EAdV2 infections are widely prevalent in the horse population tested and that SN is currently the most suitable assay for the detection of EAdV1 and EAdV2 antibodies in equine serum.