Corneal transplantation is a safe, reliable method of restoring visual acuity in patients with corneal disorders. Although it has a very high success rate, rejection can still occur, especially if the site is infected. Therefore, seeking to find better ways to manage infection risk, this study investigated a new technique, based on multiplex polymerase chain reaction (mPCR), to identify pathogens, including viruses, bacteria, and fungi, in corneal transplantation recipient sites, donor corneas and the donor cornea storage solution. The subjects comprised 50 patients who underwent corneal transplantation at Tohoku University Hospital between July 2014 and April 2015. We obtained extracted (recipient) cornea samples in 37 cases, donor cornea samples in 50 cases, and corneal storage solution samples in 50 cases (18 of these 50 samples contained DNA). Herpes simplex virus type 1 DNA was detected in four recipient corneas, Parvovirus B19 DNA was detected in two recipient corneas, Human herpes virus type 6 was detected in two donor corneas, and Aspergillus DNA was detected in one corneal storage solution sample. Thus, mPCR successfully identified pathogenic DNA in corneal tissues and storage solution, suggesting that evaluation with mPCR may improve the ability to predict the risk of infection after corneal transplantation.