Purpose: Internal cervical os (ICO) stiffness is related to menstrual pain, a key symptom of endometriosis. The study evaluated whether women with endometriosis have a stiffer ICO than unaffected women.Methods: A retrospective cross-sectional analysis was conducted using prospectively collected data from women with and without endometriosis, spanning from June 2020 to September 2022. Endometriosis was diagnosed through clinical and ultrasound evaluations, with histological confirmation in a subset of participants. Strain elastography (SE) was employed to measure tissue elasticity in four cervical regions of interest: the ICO and the anterior, posterior, and middle cervical compartments (ACC, PCC, and MCC, respectively). Tissue elasticity was quantified using a color-based scoring system ranging from 0.1 (blue, indicating less elasticity) to 3.0 (red, indicating greater elasticity).Results: Overall, 287 women were included, with 157 diagnosed with endometriosis and 130 controls. On SE, women with endometriosis exhibited a lower color score (mean±standard deviation), indicating lower elasticity, for the ICO (0.56±0.28 vs. 0.70±0.26, P=0.001) and PCC (0.69±0.30 vs. 0.80±0.27, P=0.002). Additionally, they had a lower ICO/MCC ratio (0.45±0.28 vs. 0.60±0.32, P=0.001) and ICO/ACC ratio (0.68±0.42 vs. 0.85±0.39, P=0.001). Multiple logistic regression analysis revealed that endometriosis was associated with the ICO color score (odds ratio, 0.053; 95% confidence interval, 0.014 to 0.202; R2=0.358; P=0.001), even after adjusting for confounding factors like the presence of myomas (P=0.040) and the use of hormonal therapy (P=0.001). The results were corroborated in women with histologically confirmed endometriosis (n=71).Conclusion: The findings suggest a potential relationship between a stiffer ICO and endometriosis.