Laboratory diagnosis of histoplasmosis is based on various methods including microscopy, culture, antigen and DNA detection of Histoplasma capsulatum var. capsulatum (Hcc) or H. capsulatum var. duboisii (Hcd). To improve sensitivity of existing quantitative PCR assays, we developed a new reverse transcriptase qPCR (RTqPCR) assay allowing amplification of whole nucleic acids of Histoplasma spp.. and validated on suspected cases.The limit of detection was 20 copies and the specificity against 114 fungal isolates/species was restricted to Histoplasma spp.. Whole nucleic acids of 1,319 prospectively collected consecutive samples from 907 patients suspected of histoplasmosis were tested routinely between May 2015 and May 2019 in parallel with standard diagnostic procedures performed in parallel. 44 had proven histoplasmosis due to Hcc (n=40) or Hcd (n=4) infections. RTqPCR was positive in 43/44 patients (97.7% sensitivity), in at least one specimen. Nine out of 863 cases (99% specificity) were RTqPCR positive and therefore classified as possible cases. RTqPCR was positive in 13/30 (43.3%) blood tested in proven cases. A positive RTqPCR in blood was significantly associated with Hcc progressive disseminated histoplasmosis with a positive RTqPCR in 92.3% of the immunocompromised patients with disseminated disease. This new Histoplasma RTqPCR assay enabling amplification of hcc and hcd is highly sensitive and allows the diagnosis of histoplasmosis advantageously from blood and BAL.