The aim of this study was to produce reference sera against the seven serotypes of Epizootic hemorrhagic disease virus (EHDV-1, EHDV-2, EHDV-4, EHDV-5, EHDV-6, EHDV-7, and EHDV-8). In a high containment unit, seven Prim 'Holstein calves were inoculated at day 0 (D0) with the selected strains (1 EHDV serotype per calf). Blood samples (EDTA and whole blood) were periodically taken from D0 until the end of the experiment (D31). Sera were tested with two commercially available EHDV competitive ELISAs (c-ELISA). Viral genome was detected from EDTA blood samples using in-house real-time RT-PCR. Sera taken on D31 post infection (pi) were tested and characterized by serum neutralization test (SNT) and virus neutralization test (VNT) (for calibration of reference sera). Viral RNA was first detected at D2 pi in five calves. All infected animals were RT-PCR positive at D7 pi. Seroconversion was observed between D10 and D23 pi depending on the EHDV serotype. SNT and VNT have allowed to determine the neutralizing antibody titers of each serum and the potential cross-reactions between serotypes. The two c-ELISA used in this study showed similar results. The calibrated sera are now available for the serological identification of an EHDV isolated on tissue culture or to be used as positive control in seroneutralization assay.