Abstract The Opalinida comprise ciliated protists that live as intestinal endoparasites in poikilothermic vertebrates. They are classified as one group of colourless Stramenopiles, the Opalinata, along with Proteromonadida (Proteromonas and Karotomorpha). The validity of certain opalinid genera is questionable, and their genetic relationships remain unclear. In this study, we conducted complete ribosomal DNA gene sequencing of nine opalinid species (belonging to Opalina, Protoopalina, Zelleriella, and Cepedea) from China. We compared the primary sequences, secondary structures, and characteristics of short tandem repeats within the expansion segments of the sequences. Additionally, a TimeTree analysis was performed to estimate divergence times. Our findings suggest that opalinids diverged from proteromonads ~250–220 Mya, with Protoopalina being the ancient genus. There is no genetic evidence supporting the differentiation of Cepedea and Bezzenbergeria as distinct genera, and they have been synonymized: Opalina (= Cepedea) and Protoopalina (= Bezzenbergeria); new combinations of species names are listed. Opalinid rDNA sequences exhibit greater length compared with other groups owing to A-T insertions within the expansion segments. These insertions consist of short tandem repeats and appear to be associated with speciation events that led to genus differentiation.