Background: The diagnosis of subacromial pathology is limited by the poor accuracy of clinical tests for specific pathologies. The aim of this study was to estimate the diagnostic accuracy of clinical examination and imaging features for identifying subacromial pain (SAP) defined by a positive response to diagnostic injection, and to evaluate the influence of imaging findings on the clinical diagnosis of SAP. Methods and Findings: In a prospective, diagnostic accuracy design, 208 consecutive patients presenting to their primary healthcare practitioner for the first time with a new episode of shoulder pain were recruited. All participants underwent a standardized clinical examination, shoulder x-ray series and diagnostic ultrasound scan. Results were compared with the response to a diagnostic block of xylocaine™ injected into the SAB under ultrasound guidance using ≥80% post-injection reduction in pain intensity as the positive anaesthetic response (PAR) criterion. Diagnostic accuracy statistics were calculated for combinations of clinical and imaging variables demonstrating the highest likelihood of a PAR. A PAR was reported by 34% of participants. In participants with no loss of passive external rotation, combinations of three clinical variables (anterior shoulder pain, strain injury, absence of symptoms at end-range external rotation (in abduction)) demonstrated 100% specificity for a PAR when all three were positive (LR+ infinity; 95%CI 2.9, infinity). A full-thickness supraspinatus tear on ultrasound increased the likelihood of a PAR irrespective of age (specificity 98% (95%CI 94, 100); LR+ 6.2; 95% CI 1.5, 25.7)). Imaging did not improve the ability to rule-out a PAR. Conclusion: Combinations of clinical examination findings and a full-thickness supraspinatus tear on ultrasound scan can help confirm, but not exclude, the presence of subacromial pain. Other imaging findings were of limited value for diagnosing SAP.