Background Chlamydia trachomatis (CT) is routinely diagnosed by nucleic acid amplification tests (NAATs), which are unable to distinguish between nucleic acids from viable and non-viable CT organisms. Objectives We applied our recently developed sensitive PCR (viability PCR) technique to measure viable bacterial CT load and explore associated determinants in 524 women attending Dutch sexual health centres (STI clinics), and who had genital or rectal CT. Methods We included women participating in the FemCure study (Netherlands, 2016-2017). At the enrolment visit (pre-treatment), 524 were NAAT positive (n=411 had genital and rectal CT, n=88 had genital CT only and n=25 had rectal CT only). We assessed viable rectal and viable genital load using V-PCR. We presented mean load (range 0 (non-viable) to 6.5 log 10 CT/mL) and explored potential associations with urogenital symptoms (coital lower abdominal pain, coital blood loss, intermenstrual bleeding, altered vaginal discharge, painful or frequent micturition), rectal symptoms (discharge, pain, blood loss), other anatomical site infection and sociodemographics using multivariable regression analyses. Results In genital (n=499) CT NAAT-positive women, the mean viable load was 3.5 log 10 CT/mL (SD 1.6). Genital viable load was independently associated with urogenital symptoms - especially altered vaginal discharge (Beta=0.35, p=0.012) and with concurrent rectal CT (aBeta=1.79; p<0.001). Urogenital symptoms were reported by 50.3% of women; their mean genital viable load was 3.6 log 10 CT/mL (vs 3.3 in women without symptoms). Of 436 rectal CT NAAT-positive women, the mean rectal viable load was 2.2 log 10 CT/mL (SD 2.0); rectal symptoms were reported by 2.5% (n=11) and not associated with rectal viable load. Conclusion Among women diagnosed with CT in an outpatient clinical setting, viable genital CT load was higher in those reporting urogenital symptoms, but the difference was small. Viable genital load was substantially higher when women also had a concurrent rectal CT. Trial registration number ClinicalTrials.gov NCT02694497.