A subgenomic library created from genomic DNA of Latimeria chalumnae was screened for 28S ribosomal RNA (rRNA) clones. The resulting clone was subcloned into a Plasmid vector, and over 2 kb of the 28S rRNA region was sequenced. Sequences of 28S rRNA genes were also obtained for Rhineura floridana (Squamata), Cyprinella lutrensis (Actinopterygii), and Lampetra aepyptera (Petromyzontiformes) by cloning and/or amplification by the polymerase chain reaction. The 28S rDNA sequences were aligned for all the above species as well as for the previously published 28S rDNA sequences of the genera Mus, Rattus, and Homo (Mammalia), Xenopus (Amphibia), and Drosophila (Insecta). Phylogenetic analysis of these species (using both the insect and lamprey sequences for outgroup comparison, or using only the lamprey sequence in the outgroup) produced a single optimal solution: (Outgroup(Cyprinella(Latimeria(Xenopus(Rhineura-(Homo (Rattus(Mus)))))))). Bootstrap analysis indicated that the placement of L. chalumnae on this tree was significant at p < 0.01. Previously published alternative hypotheses of relationships of Latimeria require at least 19 additional steps compared to the optimal solution; the rDNA data are sufficient to reject the hypotheses that place Latimeria in groups other than the sarcopterygians.