Objective: The diagnosis of Lyme borreliosis is based on two-tier testing using an ELISA and Western blot. About 5–10% of patients report persistent symptoms of unknown etiology after treatment, resulting in substantial difficulties in further diagnostic workup. This paper presents a study aimed at determining whether serology can differentiate between patients with persistent symptoms attributed to Lyme and other patients with Lyme borreliosis.Methods: A retrospective cohort study included 162 samples from four subgroups: patients with persistent symptoms of Lyme (PSL), early Lyme borreliosis with erythema migrans (EM), patients tested in a general practitioner setting (GP), and healthy controls (HC). ELISA, Western blots, and multiplex assays from different manufacturers were used to determine inter-test variations in PSL and to compare reactivity againstBorrelia-specific antigens among the groups.Results: In comparing the IgG and IgM reactivity by Western blot, IgG was more often positive in the PSL group than in the GP group. The individual antigen reactivity was similar between the PSL and EM or GP groups. Inter-test agreement among the manufacturers was variable, and agreement was higher for IgG testing compared to IgM.Conclusions: Serological testing is unable to define the subgroup of patients with persistent symptoms attributed to Lyme borreliosis. Additionally, the current two-tier testing protocol shows a large variance among different manufacturers in these patients.