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Background: Chlamydia trachomatis (CT), the most common bacterial sexually transmitted infection (STI), leads to pelvic inflammatory disease, infertility and chronic pelvic pain in women as well as an increased risk of vertical transmission, conjunctivitis and pneumonitis in infants. It may also be a co-factor along with human papillomavirus (HPV) in cervical cancer progression. We aimed to determine the prevalence of CT genotypes in genital specimens of women from South Khorasan, Iran and to test the association between CT and cytology statistics. Materials and Methods: This was a cross-sectional study on 248 Pap smear samples from women who visited a gynecologist for routine Pap smear testing in South Khorasan province. Nested polymerase chain reaction (PCR) was used to test the residual fluids of Pap smears for CT-DNA after cytological examination. Direct sequencing, alignment and phylogenic analyses were performed on eight samples to identify their genotypes. Results: The mean age of patients was 37.54 ± 5.21 years. Most samples had a normal cytology (214 cases, 86.29%). Overall, 31 samples were positive for CT infection (12.5%) of which 20 (9.34%) were normal and 11 (32.35%) were abnormal, with the frequency difference being significant (P=0.022). The co-infection of CT/HPV in total was identified in 14 cases (5.6%). The results of sequencing eight samples out of the 31 CT positive samples revealed the detection of genotypes D and E, each with four cases. Conclusion: We show that a high prevalence of genital CT infection is present in women with both normal and abnormal cytology; however, the higher prevalence among women in the abnormal group may indicate its involvement in cervical neoplasia. [ABSTRACT FROM AUTHOR]