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The genus Paracoccidioides comprises the species complex causing paracoccidioidomycoses (PCM). These fungi are a serious public health problem due to the long-term drug therapy, follow-up treatment, and frequent sequelae generated by the infection, such as pulmonary fibrosis. In this sense, the objective of this work was to generate bioluminescent reporter strains of Paracoccidioides spp. harboring a thermostable, red-shifted luciferase gene under the control of different constitutive promoters. The strains were generated by the integration of a species-specific codon-optimized luciferase gene upon actin or enolase promoter's control. The insertion of the constructs in Paracoccidioides brasiliensis and Paracoccidioides lutzii yeast cells were performed through Agrobacterium tumefaciens-mediated transformation. The results demonstrated the presence of several transformants harboring the luciferase gene. These transformants were further confirmed by the expression of luciferase and by the presence of the hygromycin resistance gene. Moreover, the luciferase activity could be detected in in vitro bioluminescence assays and in vivo models of infection. In general, this work presents the methodology for the construction of bioluminescent strains of Paracoccidioides spp., highlighting potential promoters and proposing an in vivo model, in which those strains could be used for the systemic study of PCM., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2022 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)