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Given the high incidence of keratoconjunctivitis in Iran (approximately 3.6–53.9%) and low effi-ciency of clinical diagnostic measures, application of laboratory tests for detection of different kerato-conjunctivitis/conjunctivitis causes and determination of their accurate prevalence is essential. In this research, conjunctival samples were collected from 100 patients with keratoconjunctivitis signs re-ferred to an eye hospital of Iran. After DNA extraction, PCR was carried out for detection of Chlamy-dia psittaci and Chlamydia felis. PCR positive products were further subjected for DNA sequencing. In this study, one sample was Chlamydia psittaci positive and none was positive for Chlamydia felis. There wasn’t a statistically significant relationship between working in the field of veterinary medi-cine or keeping a pet and Chlamydia psittaci prevalence (P>0.05). This study showed a low rate of chlamydial keratoconjunctivitis and therefore further studies for detection of other causes are neces-sary.