Choclo virus (CHOV) was described in sigmodontine rodents, Oligoryzomys fulvescens, and humans during an outbreak of hantavirus cardiopulmonary syndrome (HCPS) in 1999-2000 in western Panama. Although HCPS is rare, hantavirus-specific serum antibody prevalence among the general population is high suggesting that CHOV may cause many mild or asymptomatic infections. The goals of this study were to confirm the role of CHOV in HCPS and in the frequently detected serum antibody and to establish the phylogenetic relationship with other New World hantaviruses. CHOV was cultured to facilitate the sequencing of the small (S) and medium (M) segments and to perform CHOV-specific serum neutralization antibody assays. Sequences of the S and M segments found a close relationship to other Oligoryzomys-borne hantaviruses in the Americas, highly conserved terminal nucleotides, and no evidence for recombination events. The maximum likelihood and maximum parsimony analyses of complete M segment nucleotide sequences indicate a close relationship to Maporal and Laguna Negra viruses, found at the base of the South American clade. In a focus neutralization assay acute and convalescent sera from six Panamanian HCPS patients neutralized CHOV in dilutions from 1:200 to 1:6,400. In a sample of antibody-positive adults without a history of HCPS, 9 of 10 sera neutralized CHOV in dilutions ranging from 1:100 to 1:6,400. Although cross-neutralization with other sympatric hantaviruses not yet associated with human disease is possible, CHOV appears to be the causal agent for most of the mild or asymptomatic hantavirus infections, as well as HCPS, in Panama.