Diagnostic testing for Lyme disease (LD) remains dependent on detection of antibodies to LD using serologic assays, in adherence to the standard two-tiered testing (STTT) algorithm. We present the first analytic evaluation of the automated B31 ViraChip IgM and IgG microarray immunoblot (MIB) assays (Viramed Biotech AG, Planegg, Germany) in comparison to two different, semiautomated blot assays for LD, including the B31 ViraStripe IgM and IgG line immunoassays (LIAs) (Viramed) and the MarDx IgM and IgG Western blot (WB) assays (Trinity Biotech, Carlsbad, CA), using prospectively collected sera ( = 411) and archived, clinically characterized samples ( = 91). We show comparable overall agreement (>84%) of the ViraChip MIB assays against the two aforementioned LD blot methods. The ViraChip MIB assays were also compared to a consensus standard, whereby samples were classified as positive or negative for IgM or IgG to if the analyte-matched ViraStripe LIA or MarDx WB assay were positive or negative, respectively. The ViraChip IgM and IgG MIB assays showed>93% positive, negative, and overall agreement versus these consensus criteria. The ViraChip MIB assays were associated with a time savings of 28 min to process one full batch of samples compared to the time required for the ViraStripe LIAs. The ViraChip MIB assays can be programmed and performed on an open-system, automated enzyme-linked immunosorbent assay (ELISA) processor, negating the need for assay-specific equipment and enabling laboratories to consolidate LD testing onto a single platform. We conclude that the ViraChip IgM and IgG MIB assays may be added to the repertoire of supplemental, second-tier blot testing systems for diagnosis of LD.