Leptospirosis becomes health problem in Indonesia. Until April 2014, cases of leptospirosis transmitted by rats were reported in Boyolali with case fatality rate (CFR) 83.3%. Leptospira genus consists of various serovars and genetic types living in different environment. Classification of Leptospira species based on rpoB gene could be used as this gene has high level of polymorphism. This study aimed to identify Leptospira serovars in rat population using kinship analysis based on rpoB gene polymorphism and to describe spatial distribution of rats with Leptospira positive in Boyolali District. A cross sectional study was conducted on April 2014 at Sindon Village in Ngemplak Subdistrict and Jeron Village in Nogosari Subdistrict, Boyolali District. Polymerase Chain Reaction test was performed on 104 rat kidney samples from the two locations of study. Spatial analysis was conducted to map distribution of rats with Leptospira positive. There were six positive rpoB gene samples in Rattus tanezumi, Rattus argentiventer and Suncus murinus. Five of six positive samples showed the closest genetic kinship to Leptospira borgpetersenii serovar Sejroe based on rpoB gene. One isolate did not have a close genetic kinship to any serovar included in the cluster. Spatial analysis based on home range buffer zone showed that rats with Leptospira positive were found in 30 meter and 150 meter from leptospirosis patients.