The aim of this study was to assess the diagnostic ability of magnetic resonance imaging (MRI) in subacromial impingement syndrome (SIS), using a physiological standard of reference. MRI of the rotator cuff (RC) and subacromial injection test (SIT), a reference standard for SIS diagnosis, were performed in 125 painful shoulders. MRI diagnostic accuracies were determined using a 2 × 2 table and the percentage values of SIS diagnosis in patients with the three Zlatkin MRI stages were determined. Shoulder function was evaluated using the Constant Scale, and results were compared for stages. The sensitivity, specificity, accuracy, positive and negative predictive values of MRI for SIS diagnosis were 98.85%, 36.84%, 80%, 78.18% and 93.33% respectively. Of the 32 patients with Zlatkin stage 1 changes in MRI, 20 (62%) had SIT approved SIS diagnosis, while 47 (79%) of the 59 patients with Zlatkin 2 and all of the 19 (100%) patients with Zlatkin 3 changes were diagnosed with SIS by SIT. Mean Constant scores were 78.04 ± 18.3, 65.0 ± 19.9 and 54.52 ± 20.7 in patients with Zlatkin stages 1, 2 and 3, respectively (p < 0.05). The MRI of RC did not prove to be an excellent tool for SIT based SIS diagnosis, with its low specificity. However, the technique can give important clues, as its sensitivity and negative predictive values are high.