Abstract: Three arthropod-borne alphaviruses, western equine encephalitis viruses (WEEV), eastern equine encephalitis viruses (EEEV) and Venezuelan equine encephalitis viruses are the aetiological agents of a sometimes severe encephalomyelitis in equines and humans in the New World. With regard to the different ecology and epidemiology of these viruses, a method applied in serological screening should be able to distinguish between them as well as other related members of the genus Alphavirus in the American continent. However, this has been hampered in the past by (a) the close antigenic relationship between alphaviruses in traditional serological assays, especially in the routinely used haemagglutination-inhibition, and (b) the need of biosafety level 3 facilities to grow the viral antigens. An epitope blocking assay using an EEEV glycoprotein E1-expressing recombinant Sindbis virus and virus-specific monoclonal antibodies (mAbs) binding to the E1 of EEEV (strain NJ/60) and the E1 of Sindbis virus was established using automated flow cytometry. The test was evaluated using sera of infected and vaccinated rabbits. A cut-off value of 30% inhibition for antigenic complex-specific seroconversion was found to be sufficient for the detection of the respective infection. By using three different mAbs in parallel, we were able to detect alphavirus genus-, EEEV- and WEEV-complex-specific serum antibodies. As this test is based on the inhibition of binding of virus-specific mAbs, sera of every origin other than mouse can be tested. Thus, this assay may prove useful in the serological screening of a variety of animal species during an outbreak investigation.