The class Scyphomedusae currently comprises 5 orders, 19 families, and about 202 species. These jellyfishare important pelagic predators at the center of concern about the “rise in gelatinous zooplankton” and degradationof coastal ecosystems. Previous phylogenetic relationship studies among Scyphomedusae species reliedprimarily on morphological data. However, morphology-based phylogeny and classification of this group are difficultdue to the high diversity of morphological characters, and the topic remains controversial. Large molecularlevel advancements have yielded interesting results among the Scyphomedusae taxa. Nemopilema nomurai,Rhopilema esculentum, Cyanea nozakii, and Aurelia sp. are four common macro-jellyfish species in the Bohai andYellow seas. In this study, the nuclear ITS-5.8S rDNA gene fragments from polyps, ephyrae, and adults of thesespecies were amplified using polymerase chain reaction techniques. Homologous sequences of other Scyphomedusaejellyfish from GenBank were also included in this study. The genetic information indices, including basecomposition, sequence variation, and Kimura two-parameter genetic distance were examined. Phylogenetic treeswere constructed based on the maximum-likelihood and Bayesian inference methods to determine the phylogeneticrelationships among Scyphomedusae jellyfish. Significant length variations in the ITS-5.8S rDNA region wereobserved (length, 675–833 bp). A total of 886 aligned base pairs, with 507 variable sites and 197 parsimony informativesites, were detected in the four jellyfish species. Inter-individual internal transcribed spacer region(ITS)1 polymorphisms were observed in most of the Scyphomedusae jellyfish species(four families and nine species).Repeats in microsatellite ITS loci play an important role in length heteroplasmy. The genetic distancesamong families, species, and individuals were 0.295–0.491, 0.024–0.812, and 0–0.099, respectively. The phylogenicanalysis showed that the topologies of the maximum-likelihood and Bayesian inference trees were somewhatdifferent and different from morphological taxonomy. In conclusion, ITS is an appropriate barcoding tool to identifyjellyfish species and classify young jellyfish. Furthermore, ITS1 was highly divergent among different Scyphomedusaejellyfish and could be an appropriate marker for molecular systematic studies at the species level.Additional in-depth studies with more samples are needed to further clarify the position of Scyphomedusae jellyfishin jellyfish taxonomy.