This study evaluates how far T2 mapping can identify arthroscopically confirmed pathologies in the long biceps tendon (LBT) and quantify the T2 values in healthy and pathological tendon substance. This study comprised eighteen patients experiencing serious shoulder discomfort, all of whom underwent magnetic resonance imaging, including T2 mapping sequences, followed by shoulder joint arthroscopy. Regions of interest were meticulously positioned on their respective T2 maps, capturing the sulcal portion of the LBT and allowing for the quantification of the average T2 values. Subsequent analyses included the calculation of diagnostic cut-off values, sensitivities, and specificities for the detection of tendon pathologies, and the calculation of inter-reader correlation coefficients (ICCs) involving two independent radiologists. The average T2 value for healthy subjects was measured at 23.3 ± 4.6 ms, while patients with tendinopathy displayed a markedly higher value, at 47.9 ± 7.8 ms. Of note, the maximum T2 value identified in healthy tendons (29.6 ms) proved to be lower than the minimal value measured in pathological tendons (33.8 ms), resulting in a sensitivity and specificity of 100% (95% confidence interval 63.1–100) across all cut-off values ranging from 29.6 to 33.8 ms. The ICCs were found to range from 0.93 to 0.99. In conclusion, T2 mapping is able to assess and quantify healthy LBTs and can distinguish them from tendon pathology. T2 mapping may provide information on the (ultra-)structural integrity of tendinous tissue, facilitating early diagnosis, prompt therapeutic intervention, and quantitative monitoring after conservative or surgical treatments of LBT.