Leptospirosis is a zoonosis occurring worldwide, caused by pathogenic spirochaetes Leptospira spp. The only known reservoirs for this disease so far are rodents. In most recorded cases, leptospirosis is an acute septicemic disease of domestic and wild animals, as well as humans. Due to its complex epizootiology and insufficient recorded data on domestic and wild cats as potential reservoirs of leptospirosis, a study was conducted that included a total of forty kidney samples, of which eighteen kidney samples were of wild cats and twenty-two kidney samples were of domestic cats. In addition to the undiscovered finite number of animal species as reservoirs of leptospirosis, this disease is characterized by convalescent carrier-state, large divergences of the clinical symptoms and long-term maintenance of bacteria in the soil, vegetation and water, which represents an additional problem in the diagnosis of the disease and the collection of all necessary data on the reservoirs. The collected kidney samples were homogenized during the seven-month research period, their DNA was isolated using the NucleoSpin® Tissue method, and after the preparation of the main mixture required for conducting real-time polymerase chain reaction (RealTime PCR), every single sample of the isolated DNA was mixed with the prepared mixture, and as such was ready for analysis in the Rotor-Gene Q PCR machine. After DNA isolation and sample processing using the RealTime PCR molecular method, it was determined that not a single sample of the wild cat kidney was positive, meaning no Leptospira spp. was discovered in samples. Only one domestic cat kidney sample was tested positive. Although the obtained negative results refute the fact that wild cats could potentially, in addition to rodents, due to their lifestyle and predatory tendencies, to be reservoirs of Leptospira spp., this hypothesis needs to be further investigated, taking into account larger population samples and a wider geographical distribution of potential reservoirs. The evidence of Leptospire spp. in a kidney sample of a domestic cat implies that they could be the potential reservoir of this disease, although it was not confirmed by this research based on only one positive sample.