The widespread use of attenuated Newcastle disease virus (NDV) vaccines in poultry and the extensive presence of virulent endemic NDV isolates (NDVs) in chicken farms and in wildlife birds make the most possible existing of unnoticed mixed infections in field samples likely.However, limited papers relating to different NDV genotypes co-infection in nature were reported.The field NDV isolate was recovered from a layer farm with NDV clinical signs in Shandong in 2011, purified by chicken embryo fibroblast cell (CEF) plaque, propagated in SPF embryonated eggs, the pathogenicity characters including ICPI, MDT and IVPI were conducted as described in the OIE manual and sequenced.Nucleotide sequence editing, analysis, prediction of amino acid sequences and alignments were performed.Purifying NDVs by plaque in CEF displayed that there were at least two kinds of plaques whose genome segments late suggested that one belong to genotype Ⅶ and the other to genotype Ⅱ in view of fusion (F) and hemagglutininneuraminidase (HN) gene.Furthermore, the pathogenicity tests showed that one was a virulent strain, whereas the other was a lentogenic strain.Evidences of two NDV genotypes simultaneously infecting the same layer were found.This was one of the fewest reports of coexistence of multiple genotypes of NDV isolated from individual sick layer with significant NDV symptoms during natural infection.Those results reinforce the importance of purification by plaque cloning due to the presence of mixed infections.