Currently, no guidelines exist to help in the selection of Locator abutments for implants at different heights.The purpose of this in vitro study was to evaluate the effect of the differential heights of pairs of Locator abutments on the retention of overdentures after 6 months of simulated function.In vitro testing was performed with 4 sets of average-sized edentulous mandible analogs with 2 implants placed in the canine positions. There were 10 specimens in each of the 4 groups, with a total sample size of 40. Four groups of 2 implant-retained overdentures were fabricated, with Locator attachments at different vertical levels with differences of 0, 2, 4, and 6 mm. The overdentures were subjected to simulated function for a period corresponding to 6 months of clinical service and then tested with a universal testing machine for changes in peak load-to-dislodgement. The data were analyzed using 1-way ANOVA followed by the Tukey honest significant differences test (α=.05).Varying the heights of Locator abutments had a statistically significant effect on the retentive values of the pink Locator attachments after 6 months of simulated function (F=7.342, P=.001). The peak load-to-dislodgement ranged from 32.3 N (95% confidence interval [CI]: 26.0 to 38.6) for group 0 mm to 53.6 N (95% CI: 46.3 to 60.8) for group 6 mm. When the difference in Locator abutment heights was 2 and 4 mm, the peak load was 37.1 N (95% CI: 32.3 to 42.0) and 41.9 N (95% CI: 31.2 to 52.7). Statistical analysis revealed that the retention of group 0 mm and group 2 mm was significantly lower than group 6 mm. The retention of group 4 mm was not significantly different from groups 0 mm, 2 mm, or 6 mm.Although significant differences were found among the groups, these differences were small and may not be clinically detectable.