Sequence analysis of the hypervariable internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) is commonly used to gain insights into plant and animal population structure and phylogeny. We characterized ITS1, ITS2, and the 5.8S coding region of 18 senita (Lophocereus) individuals from 12 different populations in Baja as well as from closely related cactus species. Analyses of multiple clones demonstrated extensive paralogy in the senita rDNA gene family. We identified at least two putatively non-recombining rDNA operons in senita as well as multiple paralogous sequences within each operon. Usage of PCR, reverse transcriptase (RT)-PCR, Southern blot, primary sequence analyses of the 18S rDNA gene, and secondary structure analyses of the 5.8S rRNA showed that one of the operons encodes rDNA pseudogenes in a low copy-number (Truncated), whereas the second operon encodes an expressed rRNA (Functional). Surprisingly, we found extensive paralogy not only in the ITS regions but also in the 5.8S coding regions in senita both within and between operons. Phylogenetic analyses suggest that the second rDNA operon originated prior to the divergence of Lophocereus. A significant (p < 0.05) divergence-rate acceleration was found in the Lophocereus 5.8S rDNA coding region in the Functional operon in comparison to Pereskiopsis porteri (Cactaceae) and Portulaca molokiniensis (Portulacaceae) with Silene dioica and Spinacia oleracea as the outgroups.