Fritziana is a genus of phytotelm-dwelling frogs that inhabits bamboo and bromeliads in the southern and southeastern portions of the Brazilian Atlantic Forest. Currently, five species are known, but the existence of undescribed species has been acknowledged. Here, we conducted a species-delimitation analysis of Fritziana using deoxyribonucleic acid sequences comprising two fragments of a mitochondrial gene (16S). The species delimitation analysis (with 16S) with Bayesian inference and maximum parsimony supported the recognition of a new species as an exclusive lineage, sister to all other Fritziana species. Bioacoustic and morphological evidence also support this finding. The new species occurs from 1166 to 2146 m above sea level and inhabits bromeliads from the National Park of Serra dos Órgãos, in the municipality of Teresópolis, state of Rio de Janeiro, Brazil. Compared with its congeners, the new species has nostrils narrowly separated, diameter of tympanum equal to or larger than diameter of disc on finger III, subarticular tubercles divided, and venter uniformly beige; advertisement call with notes composed of more than two pulses, and without the longer first pulse group.