A single pair of consensus primers in the polymerase chain reaction (PCR) amplified a conserved region of the small genome segment of twenty hantavirus isolates. Isolates tested included representatives of the four recognized hantaviruses, Hantaan, Seoul, Puumala and Prospect Hill, as well as isolates from Mus musculus (Leakey), Bandicota indica (Thailand 749), and Suncus murinus (Thottapalayam). Viruses from the Nairovirus and Phlebovirus genera yielded negative results. The amplification products were 281-nucleotide pairs (np) in length, with the exception of Thottapalayam, which had an amplification product of approximately 320 np. Products of all isolates were detected by Southern hybridization with a 32P-labeled Hantaan 76—118 amplicon, while an oligonucleotide probe to a conserved region of the amplified fragment failed to detect some isolates of Seoul and Puumala viruses. Restriction endonuclease analysis allowed three groupings: Hantaan-like viruses, Seoul-like viruses, and a diverse group of patterns for the other viruses. Differences were found within the Seoul-like virus group by this method, whereas the Hantaan-like viruses were shown to be similar. RNA extracted from tissues of seropositive and seronegative rats trapped in Baltimore showed the practical application of the test. Hantavirus-specific RNA was detected in 12 (92%) of 13 seropositive rats, but not in seronegative rats. This simple method for detecting and characterizing hantaviruses has potential for epidemiologic studies and for diagnosing human hantavirus infections.