Abstract Objective Accurate prediction of spontaneous preterm birth (sPTB) is essential to target interventions. Fetal fibronectin (fFN) is a leading predictor. A quantitative fFN (qfFN) test has improved prediction, based on high vaginal swabs (HVS). It is not known how endocervical (ECS) fFN levels compare, or which has the best predictive value. Our principal aim was to determine the difference in fFN concentration between HVS and ECS and compare their ability to predict sPTB. Methods Asymptomatic high risk women (18(+0) - 30(+0) weeks' gestation) had secretions sampled from the endocervix (ECS) and vaginal fornix (HVS), analysed by the quantitative fFN analyzer (Hologic). Mean concentrations were compared; ROC curves were calculated using area under the curve (AUC) for prediction of delivery <30 and 37 weeks'. Results Mean HVS value was lower than ECS (80ng/ml (SD142) vs 217 (SD 212) (p<0.05). Predictive ability was similar: AUC of 0.92 and 0.94 respectively for prediction of sPTB <37 and 0.84 and 0.82 for <30 weeks'. Conclusions Endocervical qfFN is higher than HVS and clinicians should avoid ECS if using traditional thresholds. ECS and HVS are both useful predictors, but require different thresholds values. Further work is needed to determine whether ECS is a better and safe test to justify the difficulty in sampling.