Purpose: Using images in the facial image comparison process poses a challenge for forensic experts due to limitations such as the presence of facial expressions. The aims of this study were to analyze how morphometric changes in the face during a spontaneous smile influence the facial image comparison process and to evaluate the reproducibility of measurements obtained by digital stereophotogrammetry in these situations. Materials and Methods: Three examiners used digital stereophotogrammetry to obtain 3-dimensional images of the faces of 10 female participants (aged between 23 and 45 years). Photographs of the participants' faces were captured with their faces at rest (group 1) and with a spontaneous smile (group 2), resulting in a total of 60 3-dimensional images. The digital stereophotogrammetry device obtained the images with a 3.5-ms capture time, which prevented undesirable movements of the participants. Linear measurements between facial landmarks were made, in units of millimeters, and the data were subjected to multivariate and univariate statistical analyses using Pirouette ® version 4.5 (InfoMetrix Inc., Woodinville, WA, USA) and Microsoft Excel® (Microsoft Corp., Redmond, WA, USA), respectively. Results: The measurements that most strongly influenced the separation of the groups were related to the labial/buccal region. In general, the data showed low standard deviations, which differed by less than 10% from the measured mean values, demonstrating that the digital stereophotogrammetry technique was reproducible. Conclusion The impact of spontaneous smiles on the facial image comparison process should be considered, and digital stereophotogrammetry provided good reproducibility.