Ticks are haematophagous ectoparasites and important vectors of pathogens, including Rickettsia spp., causative agents of rickettsial diseases. The aim of this study was to monitor the occurrence of Rickettsia spp. in ticks collected from zoological gardens in the Czech Republic and Slovakia. Additionally, selected risk factors (tick developmental stage, sites within the zoo, year and season) influencing prevalence were evaluated to assess potential epidemiological implications for zoo animals and visitors. Ticks were collected from 2023 to 2025 in four zoological gardens (Brno, Ostrava, and Jihlava in Czech Republic and Košice in Slovakia) using the flagging method. Five sampling sites were selected within each zoo, with sampling performed monthly from March to November. A total of 4,960 Ixodes ricinusticks were collected and examined (Ostrava 2,095; Jihlava 1,589; Košice 720; Brno 556). Adults were analysed individually; larvae and nymphs were pooled (3 per sample), yielding 2,782 samples. DNA isolation was performed by mechanical disruption and alkaline hydrolysis (1.25% NH4OH). Rickettsia spp. was detected using PCR PCR targeting the gltA gene. Positive samples (n = 267) were subjected to sequencing. The overall minimum infection rate (MIR) was 7.2 % (358/4,960). Positivity was the highest in Košice (12.6 %, 91/720), followed by Brno (8.3 %, 46/556), Ostrava (8.2 %, 172/2095), and Jihlava (3.1 %, 49/1589), with significant differences among zoos (p<0.001). Interannual differences were observed in all zoos. In Jihlava and Košice, positivity varied also by season, developmental stage, and sites. Tick activity peaked in spring in Brno and Jihlava (57 % and 50 %) and in summer in Ostrava and Košice (42 % and 55 %). Of 358 positive samples, 223 (62.3%) were successfully sequenced. Most were Rickettsia helvetica (92.4 %), followed by Rickettsia monacensis (7.2%) and one sample matched to Rickettsia conorii/ Rickettsia slovaca. The study provides a long-term overview of Rickettsiaspp. in zoo environments and indicates that zoological gardens may act as important foci for their circulation, highlighting the need for continuous monitoring due to potential risks to animals and humans. We would like to thank the zoological gardens for their cooperation and for allowing us to collect ticks This study was supported by the Internal Grant Agency of the University of Veterinary Sciences Brno (212/2025/ FVHE).