| Summary: | Members of the <i>Borrelia burgdorferi</i> sensu lato (Bbsl) complex are etiological agents of Lyme disease (LD), and <i>Borrelia miyamotoi</i> is one of the relapsing fever <i>Borrelia</i> (RFB). Despite the serological evidence of LD in Malaysia, there has been no report from Sarawak, Malaysian Borneo. Thus, this study aimed to detect and characterize <i>Borrelia</i> in rodents and <i>Ixodes</i> ticks from primary forests and an oil palm (OP) plantation in Sarawak. <i>Borrelia yangtzensis</i> (a member of the Bbsl complex) was detected in 43.8% (14/32) of <i>Ixodes granulatus</i>; most of the positive ticks were from the OP plantation (13/14). Out of 56 rodents, <i>B. yangtzensis</i> was detected in four <i>Rattus</i> spp. from the OP plantation and <i>B. miyamotoi</i> was detected in one rodent, <i>Sundamys muelleri</i>, from the primary forest. Further, the positive samples of <i>B. yangtzensis</i> were randomly selected for multilocus sequence analysis (MLSA). The MLSA results of successfully amplified tick samples revealed a clustering with the sequences isolated from Japan and China. This study is the first evidence of <i>B. miyamotoi</i>, a known human pathogen in Malaysia, and <i>B. yangtzensis</i>, which is circulating in ticks and rodents in Sarawak, Malaysian Borneo, and presenting a new geographical record of the <i>Borrelia</i> spp.
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