Zoonotic microorganisms are a risk to human and animal health. They are often transmitted by ticks, which are important vectors. The aim of this study was molecular detection of Borrelia burgdorferi s.l., Anaplasma phagocytophilum, Ehrlichia chaffeensis, Coxiella burnetii, Francisella sp. Leptospira sp., Babesia sp. and Toxoplasma gondii. Their prevalence in ticks (Ixodes ricinus) was monitored at four localities in Moravia in the period from 2017 to 2019. At three locations in Brno, specifically in the city park Pisárky (2019), at locality near to Brno reservoir (2018) and the third area Brno – Líšeň (2017-2018). Prevalence was monitored also in Uherský Brod – Havřice (2019). Ticks were examined for the presence of zoonotic microorganisms using the conventional, multiplex or nested PCR. A. phagocytophilum was represented at the localities Brno – Pisárky (2,8 %), Brno reservoir (1,45 %) and Uherský Brod - Havřice (1,8 %). B. burgdorferi s.l. was represented at the localities Brno – Pisárky (7,2 %), Brno reservoir (5,32 %), Brno – Líšeň (36,11 %) and Uherský Brod - Havřice (3,9 %). C. burnetii was identified only at the locality Brno – Pisárky (0,3 %). E. chaffeensis was identified at the localities Brno reservoir (0,4 %) and Uherský Brod - Havřice (0,9%). Representatives of the Francisella genus were found at the Brno reservoir (1,05 %) and Uherský Brod – Havřice (0,3 %). The prevalence of microorganisms Leptospira sp., Babesia sp. and T. gondii at the locality Brno - Líšeň was examined. Their prevalence was 5,56 %, 2,78 % and 8,33 % respectively. The results of our study show that there is the potentional risk of transmission of zoonotic microorganisms at these localities. The summary results are presented in the poster section.