The uniqueness of the human dentition (UHD) is an important concept in the comparative process in bitemark analysis. During this analysis, the incisal edges of the suspects' teeth are matched with the bitemarks collected from the victim's body or crime scenes. Despite playing an essential part to exclude suspects, the UHD contained in the involved incisal tooth edges remains an assumption on bitemark level. The present study was aimed, first, to investigate three-dimensionally (3D) the UHD within different quantities of dental material from the incisal edges; second, to test these outcomes in a bidimensional (2D) simulation. Four-hundred forty-five dental casts were collected to compose 4 study groups: I – randomly-selected subjects, II – orthodontically treated subjects, III – twins and IV – orthodontically treated twins. Additionally, 20 dental casts were included to create threshold groups on subjects from whom the dental impressions were taken at 2 different moments (Group V). All the dental casts were digitalized with an automated motion device (XCAD 3D® (XCADCAM Technology®, São Paulo, SP, Brazil). The digital cast files (DCF) were integrated in Geomagic Studio® (3D Systems®, Rock Hill, SC, USA) software package (GS) for cropping, automated superimposition and pair-wise comparisons. All the DCF were cropped remaining 3 mm (part 1), 2 mm (part 2) and 1 mm (part 3) from the incisal edges of the anterior teeth. For a 2D validation, slices of 1 mm, not including incisal edges (part 4), were also cropped. These procedures were repeated in Group V, creating specific thresholds for each of the study parts. The 4 study groups were compared with its respective threshold using ANOVA test with statistical significance of 5%. Groups I, II and III did not differ from the corresponding threshold (Group V) in all study parts (p > 0.05). Scientific evidence to support the UHD was not observed in the current study. Bitemark analysis should not be disregarded but considered carefully when the suspects present similar dental alignment and morphology, such as in orthodontically treated subjects and twins, respectively.