Newcastle disease (ND) is one of the most important infectious viral diseases of poultry in Eritrea.Diagnosis of ND virus infection in Eritrea has been mainly based on clinical examination and post mortem lesions. This study describes ND diagnosis using reverse transcription polymerase chain reaction PCR (RT-PCR), virus isolation and serological techniques (haemaglutination inhibition test) from two times vaccinated 40 days old chicks in Keren poultry farm. Generally clinical signs and postmortem lesions were characterized by rapid onset, death without visible lesions, ruffled plumage, dyspnea, in appetite, weakness and green colored diarrhea. Clinical samples collected were cloacal swabs. Observed postmortem lesions were tracheal hemorrhage, hemorrhage in proventriculus especially at the junction between esophagus and proventriculus and haemorrhage in caecal tonsils. Samples collected after postmortem include spleen, intestine and caecal tonsils. High titer of specific ND virus (NDV) antibody was detected indicating the occurrence of recent infection. ND virus was isolated from aseptically collected samples using embryonated chicken eggs. RT-PCR assay described was able to detect ND virus from cloacal swabs, spleen, intestine and caecal tonsils. Therefore, the described RT-PCR assay can be adopted for rapid and confirmative detection of NDV in Eritrea. Additionally, prevention of ND should not depend only on vaccination.