Real-time polymerase chain reaction (PCR) is a quantitative method to measure the amount of amplified PCR product in real time with high sensitivity. We have applied this method to detect pathogens in cases of keratitis with an unknown cause. The scraped corneal epithelium for epithelial keratitis or aqueous humor for stromal or endothelial keratitis was obtained and DNA was extracted. The DNA from specific pathogens was amplified using specific primers and TaqMan probe, and assessed quantitatively. Here, we review previously reported noteworthy examples of keratitis diagnosed by our real-time PCR system as follows: cases with Acanthamoeba keratitis whose causative pathogen was only detected by real-time PCR despite not being detected by histological examination and culture; zoster sine herpete with atypical pseudodendrite; acyclovir-resistant herpetic keratitis estimated by changes in viral DNA copy numbers before and after treatment; and corneal endotheliitis positive for cytomegalovirus, human herpes virus-7, or human herpes virus-8. Real-time PCR helps ophthalmologists to make an early diagnosis and provide appropriate treatment for keratitis with complex clinical appearances.