Trypanosomes are blood parasites infected in various mammals, including rats. The presence of rats in human settlements can increase the chance of Trypanosoma transmission to humans. The molecular study of multispacer in Trypanosoma spp. in naturally infected rodents in Thailand is scanty. The objective of this study was to detect Trypanosoma in the blood of the captured rats in Nakhon Si Thammarat, Thailand, using microscopic and molecular techniques. This was a cross-sectional study conducted in human settlement areas. Ninety-nine blood samples were collected using cardiac puncture. A blood sample was smeared on a glass slide and examined using a compound light microscope and a scanning electron microscope. Moreover, polymerase chain reaction was applied to detect Trypanosoma evansi and T. lewisi in the blood. An additional primer set was used to confirm the species of the detected trypanosome. Approximately 18% of the rats had positive Trypanosoma infections. All Trypanosoma-positive blood samples were matched with sequences of T. lewisi. The stumpy form of trypanosome had higher nucleus related parameters than the slender form. Interestingly, the partial sequences of the alpha-tubulin gene of T. lewisi were first reported in the naturally infected RrC in this study. Based on the results obtained, T. lewisi biology, particularly the virulent components and route of transmission, pathogenesis, and in vitro experiments, are strongly recommended for further study.