Background Several symptom-relieving interventions have been shown to be efficacious among osteoarthritis (OA) patients with knee effusion; however, not every symptomatic knee OA patient has clinical effusion. Results may be over-generalized since it is unclear if effused knees represent a unique pathological condition or subset compared to knees without effusion. The primary purpose of this study was to determine if biochemical differences existed between OA knees with and without effusion. Methods The present cross-sectional study consisted of 22 volunteers (11 with knee effusion, 11 without knee effusion) with confirmed late-stage radiographic knee OA (Kellgren-Lawrence score ≥ 3). Synovial fluid samples were collected and analyzed using a custom multiplex enzyme-linked immunosorbent assay to determine eight specific biomarker concentrations (e.g., catabolic, anabolic). Results Matrix metalloproteinase (MMP)-3, tissue inhibitor of MMPs (TIMP)-1, TIMP-2, and interleukin-10 were significantly higher in the knees with effusion than in the knees without effusion. Conclusions The biochemical differences that existed between knees with and without effusion provide support that OA subsets may exist, characterized by distinct biochemical characteristics and clinical findings (e.g., effusion).