The objective of this study was to isolate and identify a hypothetical Chlamydiaceae pathogen from laying hens with an oviduct cyst, and to characterize its potential causal relation with decreased egg production. Our clinical survey showed that cystic oviducts were prevalent at rates of 10% and 15.1% in breeder and commercial hen flocks, respectively. Chlamydial antigens were detected in 20 of 50 pharyngeal swabs (40%) and in 17 of 20 oviduct tissues (85%) using enzyme-linked immunosorbent assay (ELISA) antigen detection kits. The isolated pathogen was identified as Chlamydophila psittaci via complement fixation test, PCE-ELISA, and immunofluorescence assay. Avian influenza virus, Newcastle disease virus, and infectious bronchitis virus were excluded after oviduct tissues were inoculated onto the chorioallantoic membrane of embryonating eggs. The nucleotide sequence of the omp1 gene (accession no. EF202608) from the isolate was similar to that of C. psittaci avian type C (accession no. L25436). Typical cystic oviducts were observed in specific-pathogen-free hens inoculated intraperitoneally with the isolate. The high presence of chlamydial antigen is consistent with the cystic oviducts and poor egg production. We conclude that the isolated C. psittaci is most likely associated with cystic oviducts in laying hens.