Twenty-one dengue (DEN) viruses isolated from the Caribbean (Dominica and Jamaica) during the 1981–1982 epidemic year were distinct serological and genetic variants of DEN-4 virus. These isolates were clearly identified as DEN-4 viruses using type-specific monoclonal antibodies in indirect immunofluorescence assays. However, they either were not neutralized, or were neutralized poorly using hyperimmune mouse ascitic fluids (HMAF) or rhesus monkey serum directed against the H-241 prototype strain of DEN-4 virus isolated in the Philippines in 1956. HMAF prepared against a representative Caribbean isolate, however, neutralized with similar effectiveness the homologous virus, the H-241 prototype strain, and virus strains isolated from the Pacific and Southeast Asian areas from 1973 to 1984. The Caribbean isolate exhibited no more than 30% and 16% oligonucleotide spot homology with the H-241 and Bangkok viruses, respectively, by RNA fingerprint analysis, while demonstrating 82% and 89% homology with the Gilbert and Niue Island isolates, respectively. The isolation of dengue viruses which are serologically and genetically distinct from the prototype virus emphasizes the need for continued dengue virus surveillance. The recognition of unique dengue isolates should allow the selection of reference strains and vaccine candidate strains which will induce antibodies that are equally effective in neutralizing viruses from all geographic areas.