Novel transvaginal ultrasound (TVU) markers have been proposed to improve spontaneous preterm birth (sPTB) prediction. Preliminary results of the cervical consistency index (CCI), uterocervical angle (UCA), and cervical texture (CTx) have been promising in singletons. However, in twin pregnancies, the results have been inconsistent. In this prospective cohort study of asymptomatic twin pregnancies assessed between 18+0-22+0 weeks, we evaluated TVU derived cervical length (CL), CCI, UCA, and the CTx to predict sPTB < 34+0 weeks. All iatrogenic PTB were excluded. In the final cohort of 63 pregnancies, the sPTB rate < 34+0 was 16.3%. The CCI, UCA, and CTx, including the CL was significantly different in the sPTB < 34+0 weeks group. The best area under the receiver operating characteristic curve (AUC) for sPTB < 34+0 weeks was achieved by the CCI 0.82 (95%CI, 0.72-0.93), followed by the UCA with AUC 0.72 (95%CI, 0.57-0.87). A logistic regression model incorporating parity, chorionicity, CCI, and UCA resulted in an AUC of 0.91 with a sensitivity of 55.3% and specificity of 88.1% for predicting sPTB < 34+0. The CCI performed better than other TVU markers to predict sPTB < 34+0 in twin gestations, and the best diagnostic accuracy was achieved by a combination of parity, chorionicity, CCI, and UCA.