SEN viruses (SENV) are newly discovered blood-borne single-stranded circular DNA viruses that may play a role in liver disease. To date, no serologic assays are available for the detection of SENV antigens or antibodies. We report on a rapid and sensitive molecular assay for the detection of four SENV strains (SENV-A, -C, -D, -H). This method uses PCR with universal primers and microwell capture hybridization with type-specific probes. Cut-off points to define "infected" based on chemiluminescence readings were determined from a statistical mixture model applied to samples from 300 injection drug users (IDUs) in San Francisco. Based on the estimated cut-off points, we examined the prevalence of SENV infection among 232 healthy US blood donors and assessed sensitivity and specificity of the assay in a small validation sample of infected individuals with partial sequence information.