Purpose To assess the results of a single eye bank preparing a high volume of Descemet membrane endothelial keratoplasty (DMEK) tissues using multiple technicians to provide an overview of the experience and to identify possible risk factors for DMEK preparation failure. Design Cross-sectional study. Methods setting : Lions VisionGift and Wilmer Eye Institute at Johns Hopkins Hospital. study population : All 563 corneal tissues processed by technicians at Lions VisionGift for DMEK between October 2011 and May 2014 inclusive. observation procedures : Tissues were divided into 2 groups: DMEK preparation success and DMEK preparation failure. main outcome measures : We compared donor characteristics, including past medical history. Results The overall tissue preparation failure rate was 5.2%. Univariate analysis showed diabetes mellitus ( P  = .000028) and its duration ( P  = .023), hypertension ( P  = .021), and hyperlipidemia or obesity ( P  = .0004) were more common in the failure group. Multivariate analysis showed diabetes mellitus ( P  = .0001) and hyperlipidemia or obesity ( P  = .0142) were more common in the failure group. Elimination of tissues from donors either with diabetes or with hyperlipidemia or obesity reduced the failure rate from 5.2% to 2.2%. Trends toward lower failure rates occurring with increased technician experience also were found. Conclusions Our work showed that tissues from donors with diabetes mellitus (especially with longer disease duration) and hyperlipidemia or obesity were associated with higher failure rates in DMEK preparation. Elimination of tissues from donors either with diabetes mellitus or with hyperlipidemia or obesity reduced the failure rate. In addition, our data may provide useful initial guidelines and benchmark values for eye banks seeking to establish and maintain DMEK programs.