A novel tobamovirus recently was isolated from hibiscus in Florida. Serological and molecular methods, including enzyme-linked immunosorbent assay (ELISA), dot-blot immunoassay (DBIA), tissue-blot immunoassay (TBIA), and immunocapture reverse-transcription poly-merase chain reaction (IC-RT-PCR) were compared to evaluate their usefulness for diagnosis of this virus. Each method was tested with partially purified virus preparations and tissue samples from infected hibiscus and Chenopodium quinoa plants. Indirect ELISA was more sensitive than double-antibody sandwich (DAS)-ELISA with all samples tested. The Florida hibiscus virus was detectable in hibiscus leaves and bark up to 1:12,800 and 1:6,400 dilutions, respectively, by indirect ELISA and up to 1:3,200 and 1:400 dilutions by DAS-ELISA. End-point dilutions of partially purified virus preparations from indirect and DAS-ELISA were 4 and 31 ng/ml, respectively. Florida hibiscus virus was detected by DBIA in sap from hibiscus bark and leaves at dilutions up to 1:400 and 1:800, respectively, showing that DBIA was less sensitive than either ELISA method. The virus also was detected reliably by TBIA from leaves and bark of hibiscus plants. The most sensitive method was IC-RT-PCR, which could detect as little as 500 pg/ml of virus in partially purified preparations and was 16- and 32-fold more sensitive than DAS-ELISA with hibiscus bark and leaf extracts, respectively. Over 600 hibiscus samples were tested by various combinations of these methods to validate their usefulness.