Purpose:To investigate the potential factors related to alignment instability in childhood concomitant strabismus. Design: Prospective cohort study. Methods: In total, 198 children with concomitant strabismus (57 esotropia, 141 exotropia) underwent repeat prism and alternate cover tests (PACTs) by two orthoptists who were certified by the study, and sensory tests were all performed once. We defined the alignment measurement as stable if the absolute value of the measurement difference between two orthoptists was within 10 prism diopters (PD) at distance or at near, and unstable if the difference was 10 PD or greater at distance or at near. We analyzed the relationship between the measurement variability and sensory results, patient age, and angle of deviations. Results: The mean age of the esotropia and exotropia patients was 68.5 ± 26.3 months (range, 36–164 months) and 96.1 ± 33.6 months (range, 22–200 months), respectively, and there was a significant difference in suppression related alignment instability, both at distance (P= 0.005) and at near (P = 0.039). Anisometropia also showed a significant difference at distance (P = 0.033) for alignment instability, and there was no significant statistical effect of age on measurement variability in our study. Alignment instability is positively associated with the angle of deviation, especially at distance (P = 0.019 for exotropia, P= 0.002 for esotropia). Conclusion: Clinical characteristics are proven related to alignment instability, and suppression is an important factor for alignment instability in childhood concomitant strabismus. Nevertheless, it would be entirely reasonable to consider alignment instability in relation to other factors, such as anisometropia and a large angle of strabismus, when evaluating patient deviations. Trial registration: Chinese Clinical Trial Registry, ChiCTR2200062404, Registered 4 August 2022.