Borrelia burgdorferi sensu lato, the etiological agent of Lyme disease, is tick transmitted and has a wide range of mammalian reservoirs in nature, including both wild and domestic animals. To understand the seroprevalence of B. burgdorferi s.l. in small ruminants will add value to the risk analysis of Lyme disease. The current study was intended to map the potential endemic regions of Lyme disease by large-scale investigation of sera from sheep and goats. In this study, a total of 2,758 serum samples from sheep and goats in 21 provinces located in 40 different districts of China were tested for antibodies against B. burgdorferi s.l. by enzyme-linked immunosorbent assay. The results of this survey indicated that the overall prevalence of B. burgdorferi s.l. infection ranges from 5.3 to 63.5 % (mean: 26.3%), and the infection was found in all provinces investigated. Generally, the positive rate declined from the south (34.7% in south and 32.4% in southwest) towards the north of China (18.4% in north, 16.5% in northeast and 17.2% in northwest). A significant difference was also observed in the infection rate between south and north (33.2% versus 17.4%, P<0.001). This study presents a comprehensive investigation of the serological distribution of B. burgdorferi s.l. in small ruminants in China.