Background: Lyme arthritis, a well-documented subtype of arthritis in humans and dogs, is caused by Borrelia burgdorferi and spread primarily through ticks (Ixodes spp.). While Lyme disease in horses is known to cause a variety of clinical signs, its involvement in the development of equine arthritis is controversial. As climate change enables tick populations to expand, more horses could be exposed to B. burgdorferi, therefore a greater understanding of how Lyme disease affects joints is critical to preserve soundness. Objectives: To investigate potential correlations between systemic B. burgdorferi infection and chemokine and cytokine inflammatory marker levels within synovial fluid. Study design: In vivo experiment. Methods: Synovial fluid samples were collected at the start of routine joint injection or immediately prior to surgery and analyzed for chemokine and cytokine levels. Serum samples from each horse were tested for outer surface protein (OSP) markers via Lyme Multiplex. Results: Six of twenty-five enrolled horses (24%) tested positive for at least one Lyme outer surface protein marker (OSP A 0/6; OSP C 2/6, OSP F 6/6; mean age 11.17±6.31 years), and three more horses had an equivocal result (OSP A 0/3; OSP C 1/3, OSP F 3/3; mean age 11.67±2.62 years). While most synovial inflammatory markers showed no statistical difference between systemic, serologic Lyme infection status and presumed arthritic joints (p=0.1261-0.7199) or trauma-associated joints (p=0.4560-0.9999), we observed a noteworthy negative association between CCL2 synovial fluid levels and systemic Lyme status (p=0.0142). Main limitations: Sample size was a primary limitation. Conclusions: The negative association observed between CCL2 synovial fluid levels and systemic Lyme status was also present in arthritic joints of horses with systemic B. burgdorferi infection. Notably, this contrasts with past reports of increased synovial fluid CCL2 levels associated with Lyme arthritis in other species.