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The relapsing fever group <i>Borrelia miyamotoi</i> is an emerging tick-borne pathogen. Diagnosis of infection is currently mainly based on serological methods detecting antibodies against <i>B. miyamotoi</i> glycerophosphodiester phosphodiesterase (GlpQ). Here, we scrutinized the reliability of GlpQ as a diagnostic marker and compared the seroprevalence in different study populations and by applying various immunoblotting methods. Antibodies were detected in the sera of 7/53 hunters and in 1/11 sera of Lyme neuroborreliosis patients. Furthermore, 17/74 sera of persons with high concentrations of anti-Borrelia burgdorferi sensu lato (α-Bbsl) antibodies reacted strongly with <i>B. miyamotoi</i> GlpQ in immunoblots. The <i>B. miyamotoi</i> GlpQ seroprevalence was 7/50 in α-Bbsl negative persons. In healthy blood donors from commercial suppliers and from the Austrian Red Cross, seroprevalences were 5/14 and 10/35, respectively. Strikingly, two <i>B. miyamotoi</i> PCR-positive cases from Austria had negative GlpQ serology, indicating poor sensitivity. Finally, when we analyzed sera of dogs, we found α-<i>B. miyamotoi</i> GlpQ antibody seroprevalence in tick-free dogs (<i>n</i> = 10) and in tick-exposed dogs (<i>n</i> = 19) with 2/10 and 8/19, respectively. Thus, our results indicate that GlpQ-based <i>B. miyamotoi</i> serology holds neither specificity nor sensitivity.