We examined the serological relationships among 32 hantavirus isolates collected from numerous geographic regions and hosts. We prepared rat immune sera to each virus and used these sera in hemagglutination inhibition (HAI) tests, enzyme-linked immunosorbent assays (ELISA), and plaque-reduction neutralization tests to establish the antigenic relationships among the isolates. Our studies included viruses that had been partially characterized previously, as well as isolates for which little or no serological data were available. Our results indicate that, in addition to the four established serological groups of hantaviruses, represented by Hantaan, Seoul, Puumala, and Prospect Hill viruses, there exist at least two other distinct groups of hantaviruses. These groups are represented by Thailand 749 virus, an isolate from Bandicota in Thailand, and by Thottapalayam virus, an isolate from Suncus in India. To compare more closely the antigenic relationships of the isolates to prototype Hantaan virus, we examined the reactivities of Hantaan G1- or G2-specific monoclonal antibodies with the envelope proteins of a number of the isolates. We found that several epitopes defined by monoclonal antibodies to the G2 protein were highly conserved as detected by HAI tests and ELISA. Almost all of the isolates could be neutralized by at least one G2-specific monoclonal antibody. In contrast, epitopes defined by G1 monoclonal antibodies were conserved only among Hantaan-like viruses.