Lyme-borreliosis (LB) is a tick-borne disease prevalent in many parts of the world, yet it remains largely unknown. This lack of awareness poses significant challenges for surveillance and diagnosis, requiring integrating epidemiological and clinical information. Additionally, the limited specificity and low titers of serological tests further complicate the diagnostic process. To address these issues, we investigated the molecular fine specificity of IgM antibody responses to five surface proteins of B. burgdorferi. Materials and Methods: We employed SPOT synthesis to screen for IgM epitopes and assess cross-reactivity through databank searches and ELISA. Validation was performed using ELISA and ROC curve analysis. Results: We identified ten IgM epitopes, of which four were classified as specific. The ELISA-peptide assay demonstrated a sensitivity of ≥ 87.3%, specificity of ≥ 56.2%, and accuracy of ≥ 66.6%. Two B. burgdorferi-specific epitopes, Bburg/02/huG, and Bburg/06/huG, were synthesized as polypeptides and reevaluated using ELISA with a panel of patient sera representing different pathologies. The results showed a sensitivity of 85.0% and a specificity of 100.0%, with significant differences in cross-reactivity between the leptospirosis and syphilis groups. Conclusions: These findings indicate that the identified peptide combinations could facilitate the development of new, highly specific serodiagnostic assays, thereby enhancing public health initiatives and epidemiological studies.