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There is very little scientific literature about the impact of Eel virus European X (EVEX) on eels held under artificial reproduction condition, and its effective role in eel decline is uncertain and still unclear. Following a disease outbreak of European eels, farmed in an experimental hatchery in Italy, a sample of the affected animals was analyzed to determine the cause of illness. Virological isolation, PCR assay and molecular characterization revealed the presence of EVEX in the diseased animals. The complete N, P, M and G gene regions were amplified by using novel primers specific for EVEX. The EVEX strain identified in this study was phylogenetically close to the known EVEX isolates; however, this Italian isolate diverges and localizes in a separated branch of this cluster. In addition, some unique amino acid changes in three out of four analyzed viral proteins differentiated the Italian virus from reported EVEX sequences which, although few in number, do cover different geographical areas and a 30-year time span. Molecular data are needed to gain further insight into the genetic variability of EVEX, to better define its phylogenetic and evolutionary relationships and to unravel the existence of different circulating strains. Examination of samples caught in the same area failed to detect the presence of EVEX infection. These results strongly indicate that the presence and risk factors associated with EVEX infection are not well established and further study should be performed in this field, in particular on stress and temperature dependence for disease development.