Purpose: In vitro assessment of the effect of implant site location on the stability and retention of a simulated implant-supported overdenture. Materials and methods: Study was conducted on an edentulous mandibular ridge epoxy resin model. Eight implant analogs with ball attachments were placed in positions mimicking natural dentition (canine, premolar, and molar bilaterally). Four overdenture configurations were evaluated: group I—bilateral canine and first premolar; group II—bilateral canine and second premolar; group III—bilateral canine and first molar; group IV—bilateral first premolar and first molar. Four metal hooks were attached at the location of the attachment on the polished surface of denture bases. Peak dislodging forces were measured using a universal testing machine to evaluate retention and stability. Statistical analysis was conducted using repeated measures analysis of variance (ANOVA), one-way ANOVA, and the Bonferroni post hoc method to determine specific differences between the groups. Results: Significant differences (p < 0.001) were observed when applying forces in different directions and at different implant locations. Group II (bilateral canine and second premolar) exhibited the highest mean values for vertical retention (7.85 N), anterior stability (7.17 N), posterior stability (6.99 N), and right lateral stability (4.85 N). Vertical retention and horizontal stability increased with a distal implant position up to the second premolar in simulated overdenture prostheses. Conclusion: Within the bounds of this investigation, implant distribution and position have a significant impact on the stability and retention of an implant overdenture prosthesis. Specifically, positioning implants more distally, up to the second premolar, can enhance both vertical retention and horizontal stability.