Purpose: Random dot stereoacuity can be quantified to between 40 and 800 seconds of are in preschool children by using the Randot Preschool Stereoacuity test (Stereo Optical Co, Inc, Chicago, III). To incorporate this test into clinic and research settings, the reliability of its stereoacuity scores obtained by separate examiners needs to be evaluated. The purpose of this study was to evaluate its interobserver test-retest reliability. Methods: Participants included 102 consecutive children with binocular sensory function ranging from fine to no measurable stereopsis. Clinical research participants included children with anomalous binocular vision caused by strabismus, cataracts, anisometropia, and ptosis. In a prospective study, random dot stereoacuity was measured twice under masked testing conditions by 2 examiners within a 1-hour period. Results: Interobserver test-retest reliability of the Randot Preschool Stereoacuity test is high among a population of children with diverse binocular sensory function. The correlation coefficient between individual test scores was highly significant (r = 0.97, P<.001). Mean differences between the 2 scores (0.021 log seconds of are) were not significantly different from zero (t(99) = 1.33, P >.1). The upper and lower limits of agreement were narrow, reflecting both the large sample size and the small variation between the 2 test scores. Interobserver test-retest reliability of the Randot Preschool Stereoacuity test was nearly constant across levels of functional stereoacuity, patient categorization, and age at the time of the test. Conclusions: The high agreement between the Randot Preschool Stereoacuity test scores by 2 independent observers supports its use in clinical management and research settings for the quantitative assessment of binocular sensory vision, as well as in multicentered research studies.