Background: We investigated the relationship between the mandibular Curve of Spee (COS) and a persisting primary second mandibular molar (ppM2) due to an agenesis of the second mandibular premolar, using a digital software technique. Methods: Digital dental casts were obtained from 200 patients at the Department of Orthodontics in Innsbruck and Vienna, Austria. Patients (age-, gender-, and malocclusion-matched) were equally divided into two groups (n = 100) according to the existence of a ppM2. COS depth, overjet, overbite, and angle-classification were measured digitally using the OnyxCeph3TM (version 3.2.147) software. ANOVA and Kruskal–Wallis tests were used to analyze relationships. For statistical analyses, p < 0.05 was considered as statistically significant, p < 0.01 as highly significant. Results were visualized with box plots and bar charts. Results: The deepest COS was present in patients with a ppM2. Furthermore, a positive correlation was shown between COS depth and angle-class II, between COS depth and age, as well as between COS depth and overbite. No gender differences could be observed. Conclusions: In our study population the COS depth was dependent on whether there is a ppM2 due to an agenesis of a second mandibular premolar or not, as well as on the malocclusion in sagittal direction.