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To quantitatively measure meibomian gland (MG) tortuosity in meibomian gland dysfunction (MGD) patients and normal controls and to observe the efficacy of evaluating MG tortuosity for the diagnosis of MGD. Methods: This cross-sectional study enrolled 32 obstructive MGD patients and 28 normal volunteers. Clinical assessments were performed, including symptom questionnaires, tear meniscus height, tear break-up time (TBUT), corneal fluorescein staining, lid margin abnormality, MG expressibility, and meibography. The meibomian gland tortuosity and meibomian gland density were measured by VIA software. Results: The mean age of the patients in the MGD group was 33.28 ± 9.28 years, and that of the normal controls was 25.25 ± 11.19 years. The average tortuosity of all MGs in the MGD patients was significantly larger than in the normal controls (P < 0.05). We further stratified the MGD patients into symptomatic MGD and asymptomatic groups. The average tortuosity of all MGs and of the central eight MGs was significantly higher in the symptomatic MGD patients than in the asymptomatic MGD patients (P < 0.05). Significant linear correlations were found between MG tortuosity and the lid margin score, meiboscore, meibum expressibility score, and TBUT (P < 0.05). When the diagnosis of obstructive MGD was based on the tortuosity of the central eight MGs of both eyelids, the sensitivity and specificity were 100% and 100%, respectively. Conclusions: MG tortuosity is an effective index to delineate MG morphology and to diagnose MGD, especially for the diagnosis of early-stage MGD. Translation Relevance: Calculating tortuosity quantitatively may play an important role in the diagnosis of MGD.