BACKGROUND: Clinical laxity tests are commonly used together to identify individuals with multidirectional instability (MDI). However, their biomechanical validity in distinguishing distinct biomechanical characteristics consistent with MDI has not been demonstrated. OBJECTIVE: To determine if differences in glenohumeral (GH) joint laxity exist between individuals diagnosed with multidirectional instability (MDI) and asymptomatic matched controls without MDI. METHODS: Eighteen participants (9 swimmers with MDI, 9 non-swimming asymptomatic matched controls without MDI) participated in this observational study. Participants were classified as having MDI with a composite laxity score from three laxity tests (anterior/posterior drawer and sulcus tests). Single plane dynamic fluoroscopy captured joint motion with 2D-3D joint registration to derive 3D joint kinematics. Average GH translations occurring during the laxity tests were compared between groups using an independent sample's t-test. The relationship of composite laxity scores to overall translations was examined with a simple linear regression. Differences of each laxity test translation between groups were analyzed with a two-way repeated measures ANOVA. RESULTS: Mean composite translations for swimmers were 1.7 mm greater (p = 0.04, 95% Confidence Interval (CI): 0.1, 3.3 mm) compared to controls. A moderate association occurred (r(2) = 0.40, p = 0.005) between composite laxity scores and composite translation. Greater translations for the posterior drawer (-2.4 mm, p = 0.04, 95% CI: -0.1, -4.6) and sulcus tests (-2.7 mm, p = 0.03, 95% CI: -0.3, -5.0) existed in swimmers compared to controls. CONCLUSION: Significant differences in composite translation existed between symptomatic swimmers with MDI and asymptomatic control participants without MDI during GH joint laxity tests. The results provide initial biomechanically based construct validity for the clinical criteria used to identify individuals with MDI.