Technical Abstract: Poultry in the U.S are commonly infected with avian paramyxovirus type 1 (APMV-1). Although the term APMV-1 and Newcastle Disease Virus (NDV) are often used synonymously, the Office of International Epizooties more narrowly defines NDV as only the more virulent, i.e. mesogenic or velogenic, forms of the virus. Low virulent (lentogenic) APMV-1 is common in the U.S. and is commonly used as vaccines. However, the more virulent strains of NDV, mesogenic and velogenic forms, are not normally found in the U.S. and are considered foreign animal diseases. The common term in the U.S. for these virulent viruses is exotic Newcastle Disease (END). For most foreign animal diseases, the use of serology is an extremely valuable tool, since the national herd or flock usually have no antibodies to the disease in question. However with NDV, serology is not helpful because antibody to the virulent forms of the virus cannot be distinguished from antibody due to infection with lentogenic viruses or vaccination. Although END is highly lethal for naïve poultry, clinical signs of disease are not always a reliable diagnostic tool, because vaccinated flocks may still become infected but show few signs of disease. Therefore, to successfully eradicate END virus, diagnostic tools must be available to detect and differentiate END virus from the endemic lentogenic or vaccine strains. Virus isolation in embryonating chicken eggs has historically been the primary tool for diagnosing END in the U.S., but this technique is not rapid and is dependant on the availability of embryonating chicken eggs. Recently, molecular diagnostics tools, including reverse transcriptase-polymerase chain reaction (RT-PCR), real-time RT-PCR (RRT-PCR), and sequencing of the fusion cleavage site have provided new tools for both the rapid detection and accurate differentiation of END viruses. Traditional NDV diagnostic methods will be compared to the newer molecular diagnostic techniques, and the process of validating new diagnostic tests will be examined with RRT-PCR for NDV as an example.