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To investigate the influence of periocular radiotherapy on meibomian glands.We evaluated 28 patients (40 eyes) who received radiotherapy (RT group) for conjunctival or orbital lymphoma and 30 age-matched control subjects (60 eyes). Subjects underwent slit-lamp examination of the eyelids, Schirmer test, meibography, and evaluation of tear film breakup time (TBUT), Ocular Surface Disease Index (OSDI) scores, meibomian glands evaluation (meiboscore, meibum expressibility, and lid margin abnormality scores), and tear film lipid layer thickness using an ocular surface interferometer. These parameters were compared between subjects in the RT and control groups.Meiboscores as well as meibum expressibility and OSDI scores in the RT group were significantly higher compared with those in the control group (1.6 ± 0.9 vs. 0.4 ± 0.6, 1.6 ± 1.0 vs. 0.2 ± 0.4, and 48.1 ± 21.4 vs. 6.2 ± 4.4, respectively, P < 0.001, all), whereas the Schirmer value (9.2 ± 5.1 vs. 12.3 ± 5.2, P = 0.004), TBUT (4.2 ± 2.5 vs. 6.4 ± 2.6, P = 0.001), and lipid layer thickness (61.0 ± 29.3 vs. 85.2 ± 20.0, P < 0.001) in the RT group were lower compared with those in the control group. The percentage of meibomian gland dropout was significantly correlated with age (P = 0.025) and total radiation dose (P = 0.012), regardless of the target location of irradiation. Even low-dose irradiated eyes (<30 Gy) exhibited significantly higher meiboscores (P < 0.001) and shorter TBUT (P = 0.005) compared with control eyes.Eyes that received periocular radiotherapy exhibited relatively high tear film instability induced by meibomian gland dysfunction, contributing to the high severity of dry eye symptoms.