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<h4>Purpose</h4>To explore the impact of gender mismatch on corneal allograft rejection and rejection-related graft failure in patients with repeat penetrating keratoplasty (PK).<h4>Methods</h4>A retrospective cohort was conducted at Ramathibodi Hospital, Bangkok, Thailand. Patients with repeat PK and follow-up period of at least 6 months were recruited. Demographic data, pre-operative ocular findings and donor information were collected. Patients were divided into 2 groups according to gender mismatch between donor and recipient (male to female vs others). Survival analysis was performed using the Kaplan-Meier method. The association between risk factors and the outcomes of graft rejection and rejection-related graft failure were analyzed using Cox proportional hazards regression.<h4>Results</h4>Of 68 patients with repeat PK, 26 patients (38.2%) were gender mismatched. There was no difference in 3-year rejection-free survival and graft survival between patients with gender mismatch and others (p = 0.698 and p = 0.402, respectively). Younger recipients (< 40 years) showed a significantly higher rejection rate compared to older recipients (hazard ratio; HR = 3.14, 95% confidence interval; CI 1.15 to 8.58), while patients with multiple PK (> 2 times) were found to be significantly associated with higher rejection-related graft failure compared to patients with 2 times or less PK (HR = 2.72, 95% CI 1.03 to 7.21). After multivariate analysis, only younger recipients demonstrated a statistical significance on graft rejection (adjusted HR = 2.86, 95% CI 1.04 to 7.84).<h4>Conclusions</h4>Gender mismatch might not impact corneal graft rejection or rejection-related graft failure in patients with repeat PK. Younger age was found to be a strong factor associated with graft rejection. Gender matching might not be compulsory for repeat PK.