DNA microchip used in this study was formed from miniature arrays of pseudorabies virus (PrV) gene-specific probes immobilized on a glass surface. Hybridization using DNA microchip (microarrays) was used for differentiation between virulent and attenuated PrV. The presence of four gene segments (gB, gD, gE+, and gE- ) encoding conservative glycoprotein B (gB), D (gD), and E (gE) of PrV was monitored using multiplex PCR. The amplicons were labeled with Cy5 or Cy3 dyes followed by hybridization to the gene-specific capture probes on the microchip. The presence of gD and gB, gE+ gene fragments was shown in virulent (gE+ genotype) and attenuated PrV (gE genotype), whereas gE- gene (deleted domain in gE gene) was demonstrated only in virulent, not in attenuated, virus. No cross-hybridization was observed when fluorescence labeled-PCR products of PrV were hybridized using capture probes of related viruses, such as porcine respiratory and reproductive syndrome virus (PRRSV), porcine parvovirus (PPV), Japanese encephalitis virus (JEV), and porcine circovirus type 2 (PCV-2). The assay was 10 times sensitive than gD gene-specific PCR. Overall, the results of this study suggested that the microarray might be very useful for detection and differentiation of virulent PrV from attenuated one.