Our objectives were to compare the single-line and two-line methods of cervical length measurement in the first trimester of pregnancy and to evaluate the potential value of the first trimester cervical length measured by the two methods in predicting spontaneous preterm birth. This was a prospective study in singleton pregnancies at 11+0 -13+6 weeks of gestation. Cervical length was measured by two methods: (i) a linear distance between the two ends of the glandular area around the endocervical canal (single-line method) and (ii) a sum of a linear distance from the internal os to the greatest cervical curvature and a linear distance from this point of the cervix to the external os (two-line method). The screening performance of the first trimester cervical length measured by the two different methods for the prediction of spontaneous preterm delivery was assessed by receiver-operating characteristics (ROC) curve analysis. The areas under the ROCs (AUROC) were compared by De Long test. A total of 1484 consecutive singleton pregnancies were included in this study. Spontaneous preterm delivery at <37 weeks and <32 weeks occurred in 75 cases (5.1%) and 12 cases (0.8%), respectively. The median cervical length measured by the single-line method was significantly shorter than that by the two-line method (33.5 mm vs 36.5 mm, P<0.001). Compared to the term delivery group, the median cervical length measured by the two-line method was shorter in women with spontaneous delivery at <32 weeks of gestation (36.5 mm vs 33.6 mm, P<0.01). No significant difference in the median cervical length measured by the single-line method was detected between the spontaneous preterm delivery and term delivery groups. Receiver-operating-characteristic curves demonstrated that cervical length measured by the two-line method achieved better performance in predicting spontaneous delivery at <32 weeks than the single-line method (AUROC: 0.72 vs 0.61, P<0.01). We have demonstrated that the first trimester cervical length, measured by the two-line approach, holds promise as a potential screening tool for early spontaneous preterm delivery. This article is protected by copyright. All rights reserved.