Macrophages play a key role in the inflammatory response in Lyme arthritis (LA) and could be a target for diagnosing and monitoring active Borrelia burgdorferi sensu lato (Bb) infection. Therefore, we evaluated the potential of macrophage imaging using ⁶⁴Cu-DOTATATE PET/CT for detection of Bb activity in a murine model of LA. LA was established in C3H/HeNRj mice infected with Bb B31 strain ML23 pBBE22luc. Bioluminescence imaging was performed to detect migration of spirochetes and inflammatory phagocytes to the joints. Three weeks post-infection ⁶⁴Cu-DOTATATE PET/CT imaging was performed at an early (3 h) and late (48 h) time point. Plasma levels of a systemic macrophage marker in plasma CD163 were measured. ⁶⁴Cu-DOTATATE uptake in infected joints was increased at the early (p < 0.0001) and late time points (p = 0.0005) compared with uptake in non-infected controls. No significant difference in plasma levels of CD163 was measured. ⁶⁴Cu-DOTATATE PET allows for in vivo detection and quantification of LA locally in the joints through non-invasive visualization of macrophages. In contrast, measurement of a systemic macrophage marker in plasma, CD163, did not allow to detect disease. We suggest that ⁶⁴Cu-DOTATATE PET could become a valuable diagnostic tool for in situ detection of Bb infection-related inflammation.