We investigated the use of the polymerase chain reaction for detecting genomes of herpes simplex virus, varicella-zoster virus, and cytomegalovirus from tear film of patients with clinically diagnosed herpes simplex virus keratitis. Using the polymerase chain reaction with a herpes simplex virus detection sensitivity adjusted to 1.0 plaque-forming units/ml, we detected herpes simplex virus genomic sequences in 12 of 12 epithelial keratitis specimens, two of six stromal keratitis specimens, but in none of 20 normal specimens. Neither varicella-zoster virus nor cytomegalovirus genomic sequences were detected in any sample. These results suggest that polymerase chain reaction quickly performed with reduced sensitivity is useful as a diagnostic tool for confirming clinical observations.