Background: Pathologies of the long head of the biceps (LHB) tendon are a common cause of anterior shoulder pain. While the influence of the anatomical morphology of the intertubercular groove (ITG) on the development of LHB tendon instability has been investigated with ambiguous results, the relationship of the LHB to ITG anatomy has not yet been considered in this context. The objective of this study was to reliably extract the tendon-to-groove ratio from MRI scans of symptomatic patients and examine its potential influence on the occurrence of certain causes for LHB-associated symptoms. Methods: In this retrospective study, preoperative MRI scans of 35 patients (mean age of 46 ± 14 years) presenting with anterior shoulder pain and clinical indications of LHB tendinopathy were analyzed in transversal planes. Long and short diameters of the LHB tendon and ITG were measured, cross-sectional areas of the LHB tendon and ITG were calculated from these measurements, and the ratio of cross-sectional areas (LHB/ITG) was introduced. All measurements were repeated independently by three investigators and inter-rater reliability was assessed using intraclass correlation coefficient (ICC). Thereafter, tendon-to-groove ratios were compared in patients with and without intraoperative signs of LHB tendon instability. Results: All patients exhibited intraoperative signs of LHB tendinitis, with additional findings including pulley lesions and SLAP lesions. Analysis revealed variations in the dimensions of the LHB tendon and ITG cross sections, with the tendon-to-groove ratio decreasing from 37% at the pulley to 31% at the deepest point of the sulcus. Very good inter-rater reliability was observed for all measurements. The tendon-to-groove ratio did not significantly differ (p > 0.05) in patients with or without pulley lesions or SLAP lesions. Conclusions: Our study introduced the novel parameter of the tendon-to-groove ratio of cross-sectional areas as a reproducible parameter for the description of local anatomy in the field of targeted diagnosis of LHB tendon disorders. While our findings do not yet support the predictive value of the tendon-to-groove ratio, they underscore the importance of further research with larger cohorts and control groups to validate these observations.