AIM: To determine the prevalence of ocular demodicosis by both microscopic examination and molecular detection among patients at King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok. METHODS: One hundred individuals were enrolled in the study and were divided into five age groups. The meibomian gland dysfunction (MGD) score and qualities of cylindrical dandruff (CD) were also determined. Demodex mite infestations of eyelash samples were screened by both microscopic examination and semi-nested polymerase chain reaction (PCR). RESULTS: The prevalence of ocular demodicosis as determined by microscopic examination was 42% [Demodex folliculorum (D. folliculorum) 41% and Demodex brevis (D. brevis) 1%]. Among patients who had ocular Demodex infestation, 69% have CD and had an average MGD score of 4; in patients without demodicosis, 15.5% had CD and had an average MGD score of 4.12. Prevalence of ocular demodicosis as determined by semi-nested PCR was 79% (D. folliculorum 78% and D. brevis 1%). CONCLUSION: This is the first report on the prevalence of ocular demodicosis in Thailand. Patients with CD also had Demodex mites present. Semi-nested PCR is better than microscopy for Demodex infestation detection. An extensive survey with more representative samples is required to determine the prevalence in the country.