Paracoccidioides are dimorphic fungal pathogens and the etiological agents of paracoccidioidomycosis (PCM). This severe systemic mycosis is restricted to Latin America, where it has been historically endemic. Currently, PCM presents the fewest diagnostic tools available when compared to other endemic mycoses. The main PCM diagnostic methods also have limitations. Molecular methods using different protocols have been proposed, but are restricted to a few regions. An analytical transversal study was conducted to evaluate a new molecular tool using specimens from patients diagnosed with PCM at a reference center for endemic mycoses in Rio de Janeiro, Brazil. After whole nucleic acid (WNA) extraction, RT-qPCR was performed in two independent simplex reactions, targeting the mitochondrial small subunit ribosomal RNA genes of Paracoccidioides brasiliensis and Paracoccidioides lutzii. Additionally, WNAs from all PCM-related Paracoccidioides species and from 114 other fungal strains, as well as from samples obtained from patients diagnosed with other endemic mycoses and tuberculosis, were also tested for specificity. The RT-qPCR targeting P. brasiliensis successfully amplified genetic material from all tested Paracoccidioides species but not P. lutzii, which is why a specific RT-qPCR was designed. The RT-qPCR efficiency was 1.95 (95%) with 100% analytical specificity for both targets. All included PCM clinical samples were positive (100% sensitivity) for P. brasiliensis, and all yielded negative for P. lutzii. Additionally, all samples collected from patients with other diseases were negative, reinforcing the assay's specificity. In conclusion, this study proposes a new accurate tool to cover gaps, contributing to the molecular diagnosis of this neglected disease.