Abstract Background Several Modified two-tier testing algorithms (MTTT) for Lyme disease involving sequential enzyme immunoassays (EIA) have been approved by the FDA as an alternative to conventional two-tiered testing using EIA followed by immunoblot. The MTTT algorithms are reported to be more sensitive than conventional two-tiered algorithm’s especially in early Lyme disease. The Quidel Sofia 2 Lyme assay utilizes lateral flow with fluorescence detection (FIA) providing a rapid on-demand instrument-read alternative to central laboratory batch mode testing We compared the Quidel assay as a first-tier test in an MTTT algorithm to the Zeus Scientific VlsE1/pepC10 IgG/IgM enzyme immunoassay (EIA) followed by the Zeus monovalent IgM and IgG EIA as the confirmatory test. Methods Serum specimens (n = 179) were obtained from consecutive samples sent to our clinical laboratory for Lyme serology testing. For the first-tier the samples were tested using the Zeus EIA VlsE1/pepC10 lgG/IgM test and the Quidel Sofia 2 Lyme FIA assay. Positive or equivocal first-tier tests were then tested using the Zeus monovalent IgM and IgG EIA as the confirmatory test. Results The positive percent agreement was 100%, the negative percent agreement 97.9 % with an overall agreement of 98.3%. The Kappa statistic was 0.945 (95% confidence interval 0.883–1.000 indicating “almost perfect” agreement). Conclusion The Quidel Sofia Lyme test performs well when compared to the Zeus test in a two-tiered testing MTTT algorithm. Obtaining samples to evaluate Lyme serologic tests can be challenging. Our data should prove useful for facilities considering the use of the Quidel assay.