Objective To determine the reproducibility of the mean strain value in various cervical areas and new elastographic parameters for measuring cervical stiffness evaluated by strain elastography using in vivo compression generated by internal organ movement. Methods A prospective observational study (140 singleton pregnant women; 15-33 weeks of gestation) was performed at two tertiary centers. Cervical strain was evaluated using E-cervix™ elastography. The mean strain levels of various cervical areas [internal os (IOS), external os (EOS) and endocervical area] and several new parameters [i.e. the ratio of the strain level of IOS and EOS, elasticity contrast index (ECI), and hardness ratio] were assessed twice by two independent examiners. The inter-observer and intra-observer variances were calculated using the intraclass correlation coefficient (ICC) with a 95% confidence interval (CI). Bland-Altman (B-A) analysis was also performed. Results The median gestational age was 24.0 weeks, and the mean cervical length (CL) was 3.8 cm. The intra-observer and inter-observer ICCs of the mean strain levels of the specified cervical area and new elastographic parameters were statistically significant (P < 0.001, all); the intra-observer ICC was 0.639-0.725, and the inter-observer ICC was 0.538-0.718. Conclusion The reproducibility of elastographic parameter measurements using in vivo compression is improvable.