Technical Abstract: Since the discovery of Newcastle disease virus (NDV) in 1926, nine genotypes of class I viruses and ten of class II, representing diverse and continually evolving viruses, have been described. The emergence of new virulent genotypes from global epizootics and the year-to-year changes observed in viruses of low and high virulence, implies that different genotypes of NDV are simultaneously evolving at different geographic locations, worldwide. Genomic changes occurring in virulent viruses circulating in vaccinated poultry in South America, Asia, and Africa, and laboratory experiments demonstrating shedding of challenge viruses in vaccinated animals, suggest that vaccination may play a role in the evolution of new virulent genotypes. The diversity of avian species susceptible to NDV infection and the availability of highly mobile wild bird reservoirs may favor the existence of the large genomic diversity found in low virulence NDV. Diversity and mobility create challenges for diagnostic assays predicting the need for additional rapid diagnostic tests. The possibility of Newcastle disease (ND) outbreaks, facilitated by the great mobility of birds and bird products, is a constant threat to poultry production worldwide. Constant epidemiological surveillance and pro-active characterization of circulating strains are needed to ensure that the immunological reagents, and polymerase chain reaction (PCR) primers and probes are effective in identifying representative NDV circulating worldwide. The widely used real-time reverse transcription polymerase chain reaction (RRT-PCR) matrix gene assay for identification of NDV often fails to detect low virulence viruses from waterfowl, while the RRT-PCR fusion gene assay, to identify virulent isolates, often fails to detect certain NDV genotypes. A matrix-polymerase multiplex test that identifies most of the viruses currently circulating in the U.S. and a variant of the fusion test for identification of pigeons viruses circulating in the U.S. and Europe have recently been developed. For new viruses emerging from unidentified reservoirs and with unknown sequences, recently developed random priming sequencing methods need to be incorporated into the diagnostic arsenal.