Objectives: The aim of this study was to evaluate the capacity of an intra-oral scanner (IOS) to assess the position of an endodontic guide in vitro.Methods: Fourteen extracted human teeth were placed into a maxillary model and scanned using computed tomography and a reference laboratory scanner. An ideal endodontic guide was then created and modified by adding defects of different thicknesses to simulate incorrect positions: 50 & mu;m, 150 & mu;m, 400 & mu;m, and 1000 & mu;m. For each thickness, guides were printed three times and each guide was scanned by three experimented operators using a Trios 4 IOS (3Shape, Copenhagen, Denmark). The 36 scans were compared using a best-fit alignment to the master model without defect to define the accuracy of the method and the positioning error.Results: The IOS presented a mean trueness of 1.28 & mu;m (SD= 12.70) and a mean precision of 11.52 & mu;m (SD= 62.17). Considering all sizes of defect, the mean measured position of the endodontic guide was highly correlated (R>0.99) with the expected position. Compared to the ideal guide, there was a mean linear deviation of 46.11 & mu;m (SD= 23.21) and a mean angular deviation of 5.9 degrees (SD= 1.2); this deviation was not influenced by the operator.Conclusion: The present study found that an IOS had good performance to detect a positioning error of the endodontic guide in vitro.Clinical Significance: This new application of IOS has a promising potential in clinical practice to assist practitioners during the fitting of guides.