Widely employed diagnostic antibody serology for Lyme disease, known as standard two-tier testing (STTT), exhibits insufficient sensitivity in early Lyme disease, yielding many thousands of false-negative test results each year. Given this problem, we applied serum antibody repertoire analysis (SERA), or next-generation sequencing (NGS)-based serology, to discover IgG and IgM antibody epitope motifs capable of detecting Lyme disease-specific antibodies with high sensitivity and specificity. Iterative motif discovery and bioinformatic analysis of epitope repertoires from subjects with Lyme disease ( n = 264) and controls ( n = 391) yielded a set of 28 epitope motifs representing 20 distinct IgG antibody epitopes and a set of 38 epitope motifs representing 21 distinct IgM epitopes, which performed equivalently in a large validation cohort of STTT-positive samples.