A bioinformatics approach using single-nucleotide polymorphism (SNP) analysis was performed to improve the current real-time reverse transcription–PCR (RRT-PCR) tests for the rapid detection of Newcastle disease virus (NDV). In total, 422 NDV complete genomes were analyzed using the Virus Pathogen Resource to compare the conservation of the primer and probe sequences and to select regions to develop new RRT-PCR tests. The sensitivity and specificity of the three new RRT-PCR tests targeting the nucleoprotein (NP) and polymerase (L) genes were optimized and were compared with established tests for NDV detection. The SNP analysis was also used to identify the number of mismatches between selected primers/probes and the NDV complete genome sequences. The SNP analysis, averaged over the entire primer or probe, showed the primer/probe sequences of three new tests were more conserved than the primer/probe sequences of the commonly used test targeting the matrix (M) gene. The M RRT-PCR test was compared with the new tests on a panel of 46 viruses, comprising 31 NDV isolates. Limit of detection (LOD) varied from 1.3 to 3.7 log 50% egg-infective doses using five isolates from different genotypes by all tests. The two RRT-PCR tests targeting the L and M genes detected three out of five isolates with the lowest LOD. The NP and M RRT-PCR tests had the lowest and highest rates of genetic variants, respectively, among all probes. Because currently used tests are likely to miss some isolates, the availability of validated alternative tests provides alternatives for detection of viral variants that can be rapidly deployed to diagnostic laboratories.