DNA microarrays combine high-precision technology with advanced molecular biology to achieve high-throughput screening of DNA fragments. In this study, we investigated the potential of the cDNA microarray technique to identify and discriminate PCR derived amplicons from genetically highly similar viruses. The wide range of sequence variation among hantaviruses makes them suitable as a model for this purpose. The hantaviruses, carried by rodents, cause several hundred thousand cases of severe human disease every year in many parts of the world. A hantavirus-specific microarray, including DNA fragments from 12 viral isolates of six different hantaviruses, was designed. The S and M genome segments were represented by 500-nucleotide overlapping and 250-nucleotide non-overlapping fragments. A considerable ability to distinguish between different hantaviruses was demonstrated using a novel analysis method. Even different isolates of a single virus, were identified correctly despite 90% sequence similarity. The distinction ability was accompanied by a tolerance for smaller sequence differences, which makes the microarray suitable for testing samples containing unknown viruses. Viral genetic material found in samples from the lungs of bank voles caught in the wild was identified precisely, which demonstrated further the potential for this technology. J. Med. Virol. 72:646–655, 2004. © 2004 Wiley-Liss, Inc.