The Palatal Rugae are considered a useful human identification marker for both orthodontists and forensic personnel. The principal aim of the present study was to evaluate the stability of palatal rugae with a 3D-3D superimposition procedure following Slow Maxillary Expansion (SME), in order to assess whether they kept their uniqueness and validity for human identification, even after a specific dental treatment. For this purpose, a sample of 27 digital dental models - belonging to growing patients (13 males and 14 females), aged between 8.5 and 15 years, who underwent SME therapy - was retrospectively studied and compared with a control group of 27 untreated subjects - (13 males and 14 females). Digital dental models were obtained pre-treatment and at device removal; both were processed by means of an intraoral scanner. A superimposition procedure was thus performed to reach the minimum point-to-point distance between two models of palatal rugae. Intra- and inter-observer differences were statistically analyzed by paired Wilcoxon test and Intra-class Correlation coefficient (ICC), showing values larger than 0.93. There was no difference in Root-Mean-Square (RMS) values between untreated control subjects and subjects treated with Leaf Expander (p = 0.062). A RMS value of 0.43 was the threshold to distinguish the pooled group (“Untreated” and “Leaf”) from any mismatch. According to the obtained results, this study failed to reject the null hypothesis and presented no differences between the RMS values of the Test group and the RMS values of the untreated control group. This work highlighted the usefulness of 3D superimposition procedure for purposes of human identification, in subjects undergoing dental treatment. However, keeping in sight the forensic use of this technique as a helpful probation element in court, further studies should be performed to confirm these findings.