Accurate identification of tick species is essential for understanding their role in pathogen transmission and guiding vector control strategies. Although morphological classification is widely used, it can be limited by specimen damage or immature developmental stages. In this study, we comparatively evaluated mitochondrial 16S and 12S ribosomal DNA (rDNA) markers for species-level identification and morphological verification of hard ticks and rodent-associated ectoparasites collected from vegetation, wild rodents, and patients in Gwangju, Republic of Korea. A total of 74 morphologically identified adult ticks—Haemaphysalis longicornis, Haemaphysalis flava, and Ixodes nipponensis—along with 3 damaged ticks removed from patients, were analyzed. PCR and sequencing success varied among species and markers. The 16S rDNA marker showed a higher amplification rate but lower sequencing success, whereas 12S rDNA showed more consistent performance. I. nipponensis showed the most balanced performance (89.5% for 16S and 78.9% for 12S), H. flava showed moderate variation, and H. longicornis demonstrated strong marker dependence, with low 16S but complete 12S success. Importantly, 16S rDNA identified 3 severely damaged ticks from patients—2 I. nipponensis and 1 Amblyomma testudinarium—demonstrating its diagnostic value when morphology was uninformative. Phylogenetic analyses of both markers yielded well-supported clades consistent with morphological identification. Additionally, 12S rDNA identified 3 flea specimens (Ctenophthalmus sp. and Neopsylla specialis) obtained during tick collection from wild rodents, indicating exploratory applicability to other rodent-associated ectoparasites. These findings support mitochondrial 16S and 12S rDNA markers as reliable tools for tick identification, especially when morphological assessment is limited by damage or developmental stage.