Background: Chlamydia trachomatis is one of the most common sexually transmitted infections in the world. However, there is no detailed information on its incidence, especially in developing countries where routine laboratory diagnosis is unavailable. Objectives: This study aimed to investigate the prevalence of endocervical C. trachomatis infection and related risk factors among women attending the University Gynecology Clinic of Birjand, East of Iran. Methods: This cross-sectional study was conducted on 195 women attending the University Gynecology Clinic in South Khorasan, Birjand. Endocervical sampling was performed in a lithotomy position using a sterile brush. Identification of C. trachomatis was performed by real-time PCR method using GeneProof C. trachomatis PCR kit. Data on socio-demography and potential risk factors for genital infection were analyzed using SPSS software (version 21). Results: In the study, the prevalence rate of C. trachomatis among women was reported 4.1% (8/195 subjects). Statistical analysis showed that the rate of C. trachomatis infection in women was only statistically related to the history of vaginal infection (P = 0.001). Although there was no statistically significant association between chlamydial infection and age, the highest infection rate was in women less than 30 years old. Conclusions: Given the relatively significant incidence of C. trachomatis infection among women, our findings highlight the importance of routine screening and early diagnosis of C. trachomatis to control the infection.