A loop-mediated isothermal amplification (LAMP) assay was developed to detect Borrelia burgdorferi s. l. in ticks, which is a pathogen that causes Lyme disease. Cross-reactions with Chlamydia psittaci, Mycoplasma mycoides subsp. capri and some tick-borne pathogens were excluded. Analytical sensitivity of LAMP showed its detection limit was from 0.02 to 0.2 pg of DNA in detection of the reference samples at 65°C for 40 min. The performance of LAMP was assessed by testing 110 samples from susceptible tick species and comparing the results with conventional and nested PCR tests previously described. The results demonstrated that LAMP was significantly more sensitive than the conventional PCR (32.7% versus 15.5%, P < 0.05) and slightly more sensitive, although not significantly so, than nested PCR (32.7% versus 26.4%, P > 0.05). The assay was used to analyse a total of 1052 ticks collected from eight provinces in China. The results showed that the infection rates of B. burgdorferi s. l. varied from 12.5% to 88.9% across the different geographical sites. Selected positive samples were subjected to sequencing and sequence analysis for conformation of the accuracy of the assay. Here we report a highly sensitive, specific and easy diagnostic assay based on LAMP technology. These data indicate that LAMP is a useful approach for detecting B. burgdorferi s. l. in field-collected ticks and has the potential as an alternative tool for the ecological and epidemiological surveillance of Lyme disease.