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Objectives: To investigate the presence of Demodex mites on the eyelashes of a Japanese population and to explore its associations with subjective ocular symptoms and clinical ocular surface signs, including lid margin findings and fluorescein breakup time (FBUT). Methods: Sixty-three Japanese Tokyo-based volunteers were examined (24 men and 39 women; mean±SD of age, 50.6±15.8 years). Eyelash sampling was performed by epilating three lashes from the center of the right upper eyelid. Eyelashes were tested for Demodex mites with a light microscope. The subjects completed questionnaires to assess subjective symptoms and risk factors for dry eye disease (DED) and underwent general examinations for DED, including FBUT and cornea-conjunctival staining with fluorescein. Meibomian gland function was also investigated in lid margin findings, vascularity and mucocutaneous junction movement, lid irregularity, plugging, pouting, and meibum secretion grade scores. Results: Demodex mites were found in 20.6% (13/63) of the volunteers. Lid margin vascularity and meibum grades in the upper eyelid margins were significantly associated with the presence of Demodex mites. However, there were no significant differences in the diagnosis of meibomian gland dysfunction, FBUT, or ocular surface discomfort between the subjects with or without Demodex mites. Conclusions: Demodex mites are not rare in the cilia of a Japanese population. Lid margin vascularity and lower meibum quality may be associated with the presence of Demodex mites.