Borrelia Burgdorferi is the causative pathogens for Lyme disease. A loop-mediated isothermal amplification (LAMP) rapid assay was developed for genotyping Borrelia borgdorferi based on the outer surface protein A (OspA) gene. All three different genotypic isolates including B. burgdorferi sensu stricto, B. afzelii and B. garinii, distributed mainly in China were detected and genotyped successfully using the LAMP method. The genotyping of Borrelia burgdorferi would provide evidence for treatment of patients with different clinical symptoms and for control of Lyme disease.