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Mayaro and Una viruses (MAYV, UNAV) are mosquito-borne alphaviruses that may cause an acute febrile illness characterized by headache, retro-orbital pain, and rash that may progress to a severe and prolonged arthralgia. MAYV was first isolated in Trinidad in 1954, and UNAV was first identified in northern Brazil in 1959. Since then, numerous isolates of these agents have been made from humans, wild vertebrates, and mosquitoes in several countries in northern South America. Serological evidence suggests that these viruses are also present in portions of Central America. Because little is known about the natural transmission cycle of MAYV and virtually nothing is known about UNAV transmission, 63 isolates covering the known geographic and temporal ranges were used in phylogenetic analyses to aid in understanding the molecular epidemiology. Approximately 2 kb from the E1 and E2 glycoprotein genes and the complete 3′ non-coding region were sequenced. Phylogenetic analyses of these sequences indicated that two distinct genotypes of MAYV exist with a distinct clade consisting exclusively of UNAV (previously designated as a subtype of MAYV). One MAYV genotype (genotype D) contains isolates from Trinidad and the northcentral portion of South America including Peru, French Guiana, Surinam, Brazil, and Bolivia. All of these isolates are highly conserved with a nucleotide divergence of < 6%. The second MAYV genotype (genotype L) contains isolates only from Brazil that are highly conserved (< 4% nucleotide divergence) but are quite distinct (15–19%) from the first genotype isolates. These analyses provide possible explanations for the natural ecology and transmission of MAYV and UNAV.