The performance of synovial fluid biomarker D-lactate to diagnose septic arthritis (SA) and differentiate it from crystal-induced arthritis (CA), other non-infectious rheumatic joint diseases (RD) and osteoarthrosis (OA) was evaluated. Consecutive adult patients undergoing synovial fluid aspiration due to joint pain were prospectively included in different German and Swiss centers. Synovial fluid was collected for culture, leukocyte count and differentiation, detection of crystals, and D-lactate concentration. Youden's J statistic was used to determine optimal D-lactate cut-off value on the receiver operating characteristic (ROC) curve by maximizing sensitivity and specificity. In total 231 patients were included. Thirty-nine patients had SA and 192 aseptic arthritis (56 patients with OA, 68 with CA, and 68 with RD). The median concentration of synovial fluid D-lactate was significantly higher in patients with SA than in those with OA, CA, and RD (p<0.0001, p<0.0001 and p<0.0001, respectively). The optimal cut-off of synovial fluid D-lactate to diagnose SA was 0.033 mmol/L with a sensitivity of 92.3 % and specificity of 85.4 % independent of previous antimicrobial treatment. Sensitivity and specificity of synovial fluid leukocyte count at a cut-off of 20,000 cells/µL was 81.1 % and 80.8 %, respectively. Synovial fluid D-lactate showed a high performance for diagnosing SA which was superior to synovial fluid leukocyte count. Given its high sensitivity and specificity, it serves as both an effective screening tool for SA and a differentiator between SA and RD, especially CA.