Introduction D-Dimer was recently identified as an additional biomarker in the diagnosis of hip and knee periprosthetic joint infection (PJI). Currently, there is only one study in literature dealing with the role of D-Dimer in the diagnosis of shoulder PJI. The purpose of this study was, therefore, to validate the sensitivity and specificity of D-Dimer in detecting shoulder PJI. Materials and methods All patients, who underwent septic or aseptic revision shoulder arthroplasty in our institution between November 2018 und March 2021, were analyzed. Our cohort consisted of 30 patients, of that 14 (47%) had a shoulder PJI according the last proposed criteria of the International Consensus Meeting. The diagnostic validity of serum D-Dimer regarding the detection of PJI was analyzed. Results The mean D-Dimer level was significantly higher for the patients with shoulder PJI compared to patients with aseptic failure (1.44 +/- 1 mg/l vs. 0.76 +/- 0.6 mg/l, p = 0.025). Coagulase-negative staphylococci were the most commonly isolated pathogens, in 9/14 patients (64%), followed by Cutibacterium acnes in 5/14 patients (36%). According to the ROC analysis, a serum D-Dimer threshold of 0.75 mg/l had a sensitivity of 86% and a specificity of 56% for detection of a shoulder PJI. The area under curve was 0.74. A serum C-reactive protein (CRP) cutoff of 10 mg/l showed a sensitivity of 69% and a specificity of 88%. When both serum D-Dimer and CRP above the thresholds of 0.75 mg/l and 10 mg/l, respectively, were used to identify a PJI the sensitivity and specificity were 57% and 100%, respectively. Conclusions Serum D-Dimer showed a good sensitivity but a poor specificity for the diagnosis of shoulder PJI. Combination D-Dimer and CRP led to improvement of the specificity, however, at the cost of sensitivity. Thus, combination of both methods may be used as a confirmatory test in the diagnosis of shoulder PJI but not to rule out infection.