To examine the relationship between the lower uterine segment (LUS) thickness and the onset of labor in dichorionic twin pregnancies experiencing threatened preterm labor. This prospective cohort study included dichorionic twin pregnancies between 24 and 32weeks of gestation, presenting with symptoms of preterm labor. The LUS thickness and mid-anterior wall thickness were measured via transabdominal ultrasonography, cervical length, and posterior cervical lip thickness were measured transvaginally. The presence of the cervical sliding sign and funneling findings were recorded. Among the patients with an onset of labor before the 34th week, the mean LUS thickness was 3.8±0.9 mm, compared to 4.6±1.1 mm in those with an onset of labor at or after 340/7 GW, indicating a statistically significant difference (p=0.012). Similarly, accepting the GW threshold as 350/7 weeks, a statistically significant difference was found in the mean LUS thickness, which was 4.0±1.0 mm in those with an earlier onset of labor and 4.7±1.0 mm in those with a later onset of labor (p=0.022). While univariate analysis indicated that the LUS thickness was a significant predictor (p=0.017 for <34 GW and p=0.028 for <35 GW), multivariate analysis showed a reduced effect (p=0.04 and p=0.06, respectively). LUS was significantly thinner in pregnancies with an onset of labor before the 34th and 35th GW. The measurement of the LUS thickness can be considered an alternative method for predicting spontaneous preterm birth in dichorionic twin pregnancies.