<p>Infection with the protozoan parasite <i>Trypanosoma cruzi</i> can lead to Chagas disease, and diagnosis of infection mostly relies on serological testing, although no gold standard exists. The evaluation of treatment efficacy is also challenging as seronegativization takes decades to be detected. The Multi-Cruzi platform, which provides a serological profile against 15 parasite antigens, has been proposed as promising alternative for confirmatory diagnostic and monitoring of treatment response. To further evaluate this platform, we performed epitope mapping of the Multi-Cruzi antigens with peptide microarrays, using confirmed <i>T. cruzi</i> positive samples, as well as serologically discordant samples with positive <i>T. cruzi</i> PCR. Epitope conservation among parasite strains was also assessed. We identified multiple linear epitopes among Multi-Cruzi antigens with samples from patients with confirmed serology, although some redundancy in epitopes may limit the breadth of the antibody profile evaluated. On the other hand, this antigen panel showed very limited reactivity with serodiscordant samples with confirmed <i>T. cruzi</i> infection, with weaker recognition of fewer epitopes. Epitope conservation ranged from highly conserved to more variable. The usefulness of this platform may be limited to a fraction of patients and parasite strains. The addition of alternative antigens may help improve the monitoring of treatment response of serodiscordant patients.</p>