We report two cases of louse-borne relapsing fever observed at our Institution in June 2016. Both patients were young asylum seekers from Africa who had recently arrived in Milan, Italy. Notably, direct microscopic examination of peripheral blood smears was repeatedly negative for the presence of spirochetes and the diagnosis, supported by clinical and epidemiologic evidence, required molecular confirmation by polymerase chain reaction amplification of DNA extracted from blood and sequencing of the amplified products.