Osteoglossidae, a family of ancient fishes known as “living fossils,” has high economic, cultural, and scientific value. In this study, we analyzed the molecular phylogeny of higher-order elements in the Osteoglossidae family using ribosomal DNA sequences. The complete sequences of the ribosomal gene of Arapaima gigas, Heterotis niloticus, Osteoglossum bicirrhosum, O. ferreirai, Scleropages formosus, S. jardinii, and S. leichardti, obtained using second-generation and third-generation sequencing methods, were as follows: 11714 bp, 8957 bp, 12057 bp, 11556 bp, 10377 bp, 10724 bp, and 10725 bp, respectively. The base content of GC ranged from 63.78% to 66.13%. The lengths of 18S rDNA were 1835 bp; GC content ranged from 54.06% to 54.5%. The genetic distance ranged from 0.000 to 0.024, and the sequence identity varied from 97.52% to 100%. The lengths of ITS1 rDNA were between 598 bp and 871 bp; GC content ranged from 72.6% to 78.3%. The genetic distance ranged from 0.000 to 0.852, and the sequence identity varied from 38.34% to 99.5%. The lengths of 5.8S rDNA were 159 bp; GC content ranged from 57.23% to 57.86%. The genetic distance ranged from 0.000 to 0.019, and the sequence identity varied from 98.11% to 100%. The lengths of ITS2 rDNA were between 327 bp and 612 bp; GC content ranged from 70.34% to 80.07%. The genetic distance ranged from 0.000 to 0.799, and the sequence identity varied from 31.63% to 99.53%. The lengths of 28S rDNA were between 3996 bp and 4419 bp; GC content ranged from 62.42% to 65.69%. The genetic distance ranged from 0.000 to 0.066, and the sequence identity varied from 84.98% to 99.95%. The lengths of IGS rDNA were between 1815 bp and 4679 bp; GC content ranged from 63.73% to 71.52%. The genetic distance ranged from 0.001 to 1.877, and the sequence identity varied from 19.31% to 98.31%. The phylogenetic tree was constructed using the neighbor-joining method and Bayesian inference method on the 18S, ITS1, ITS2, and 28S single sequences and the 18S+ITS1+5.8S+ITS2+28S combined sequence. The results showed that the topological structure of the two trees was consistent. Moreover, this result is concordant with the traditional classification of seven species of bony tongue fish, except for ITS1, which can be used as a molecular marker of Osteoglossidae. Our findings provide data for the phylogenetic study of ancient fishes and to enrich the diversity and evolution mode research of fish using ribosomal RNA genes.