Background and Purpose <p>The purpose was to investigate the agreement of transperineal and transvaginal cervical length (CL) and uterocervical angle (UCA) measurements in pregnant women at 16–24 gestational weeks.</p> Methods <p>Pregnant women at 16–24 weeks of gestation referred to our hospital for routine second-trimester ultrasonography were included. After informed consent and collecting baseline characteristics, all patients underwent TVUS followed by TPUS and CL and UCA were measured accordingly. Agreement was assessed using intraclass correlation coefficient (ICC) and Bland-Altman plot. Patient-reported discomfort or pain during each examination method was also documented. We also performed subgroup analysis based on BMI to evaluate the impact of BMI on ultrasound measurements.</p> Results <p>205 pregnant women at 16–24 gestational weeks were recruited, of which 5 patients were excluded due to sub-optimal image acquisition and 200 participants were included for final analysis. The mean CL measurements with the transvaginal and transperineal methods were 39.39 ± 5.6&#xa0;mm and 39.12 ± 6.33&#xa0;mm; respectively. The mean UCA measured using TVUS and TPUS were 117.35 ± 32.50<b>°</b> and 114.68 ± 35.22°; respectively. Agreement between the two was good for CL with an ICC of 0.90(95% CI, 0.867–0.924) and good for UCA with an ICC of 0.84 (95% CI, 0.793–0.881). The concordance was higher among patients with BMI &gt; 30&#xa0;kg/m², with ICC of 0.920(95% CI, 0.845–0.959) for CL and 0.932(95% CI, 0.868–0.965) for UCA.</p> Conclusion <p>TPUS demonstrated good agreement with TVUS for measuring CL and UCA during the second trimester and may be considered when TVUS cannot be performed.</p>