Lyme disease is on the rise in Canada. It is a notifiable disease, and when infection is disseminated, serological testing provides supplemental evidence to confirm a case.To describe the current diagnostic tests for Lyme disease, review the recommended approach to laboratory testing for Lyme disease and identify future research priorities for Lyme disease laboratory diagnostics in Canada.A review of the literature was carried out. We then summarized parameters to consider before Lyme disease testing is conducted, described the current best practice to use a two-tiered diagnostic algorithm for the laboratory confirmation of disseminated Lyme disease, and analyzed the advantages and disadvantages of the supplemental tests for Lyme disease.Diagnostic testing is indicated in people who have symptoms of disseminated disease and a history of exposure to vector ticks. To maximize sensitivity and specificity, a two-tiered serological approach is recommended, consisting of an enzyme immunoassay (EIA) screening test followed by confirmation with Western blot (WB) testing. A number of other diagnostic tests are available; however, these are largely for research purposes.Two-tiered serology is currently the best approach available to assist doctors when they are making a diagnosis of disseminated Lyme disease. The Public Health Agency of Canada (the Agency) will seek to improve on this approach through standardization of the Lyme disease diagnostics used across laboratories in Canada, evaluation of test performance characteristics of current and new diagnostic platforms and development of a process to secure robust serum panels to assist in the development and evaluation of new diagnostic tests for Lyme disease.