To determine the relationship between synovial biomarker concentrations and severity of lameness and to assess the ability to differentiate normal from osteoarthritic joints with synovial biomarker concentrations. Prospective clinical study. Twelve hounds with no evidence of osteoarthritis (OA) and 27 client-owned dogs with unilateral lameness and joint pain in a single joint from naturally occurring OA. Enrollment in the OA group required a history of lameness, radiographic evidence of OA on orthogonal joint radiographs, and ≥6% gait asymmetry between contralateral limbs. The concentrations of 14 synovial OA biomarkers in synovial samples obtained after gait analysis were measured with enzyme-linked immunosorbent assays and compared between normal and OA joints. Concentrations of monocyte chemoattractant protein (MCP)-1, substance P, interleukin (IL)-6, IL-8, KC-like, matrix metalloproteinase (MMP)-1, and MMP-3 were greater (P ≤ .05) in OA than in normal joints. The concentrations of bradykinin and tissue inhibitors of metalloproteinase-4 were decreased in OA compared with normal joints. Monocyte chemoattractant protein 1 was identified as the most accurate marker to distinguish OA from normal joints. No correlation was detected between any OA biomarker concentration, individually or in combination, and severity of gait asymmetry at the walk. Differences in proinflammatory and anti-inflammatory biomarkers were detected between OA and normal joints, but no relationship was identified between biomarker concentrations and gait asymmetry in dogs with OA. This information will help guide future studies to elucidate how factors such as disease chronicity, severity, and etiology affect these relationships.