Equine hepacivirus (EqHV, Flaviviridae, Hepacivirus) is a small, enveloped, RNA virus generally causing sub-clinical hepatitis with occasional fatalities. EqHV is reported in equids worldwide, but for Italy data are limited. To address this, a survey study was set up to estimate prevalence at national level and among different production categories (equestrian, competition, work and meat and reproduction) and national macro-regions (North, Central, South and Islands). Data obtained testing 1801 horse serum samples by Real-Time RT PCR were compared within the categories and regions. The NS3 fragment of the PCR-positive samples was sequenced by Sanger protocol for phylogenetic and mutational analysis. The tertiary structure of the NS3 protein was also assessed. The estimated national prevalence was 4.27% [1.97-6.59, 95%CI] and no statistical differences were detected among production categories and macro-regions. The phylogenesis confirmed the distribution in Italy of the three known EqHV subtypes, suggesting also a possible fourth sub-type that however requires further confirmation. Mutational profiles that could also affect the NS3 binding affinity to the viral RNA were detected. The present paper demonstrates that EqHV should be included in diagnostic protocols when investigating causes of hepatitis and in quality control protocols for blood derived products, due to its parental transmission.