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Accurate identification of tick-borne bacteria, including those associated with rickettsioses, pose significant challenges due to the polymicrobial and polyvectoral nature of the infections. We aimed to carry out a comparative evaluation of a non-targeted metagenomic approach by nanopore sequencing (NS) and commonly used PCR assays amplifying Rickettsia genes in field-collected ticks. The study included a total of 310 ticks, originating from Poland (44.2 %) and Bulgaria (55.8 %). Samples comprised 7 species, the majority of which were Ixodes ricinus (62.9 %), followed by Dermacentor reticulatus (21.2 %). Screening was carried out in 55 pools, using total nucleic acid extractions from individual ticks. NS and ompA/gltA PCRs identified Rickettsia species in 47.3 % and 54.5 % of the pools, respectively. The most frequently detected species were Rickettsia asiatica (27.2 %) and Rickettsia raoultii (21.8 %), followed by Rickettsia monacensis (3.6 %), Rickettsia helvetica (1.8 %), Rickettsia massiliae (1.8 %) and Rickettsia tillamookensis (1.8 %). Phylogeny construction on mutS, uvrD, argS and virB4 sequences and a follow-up deep sequencing further supported R. asiatica identification, documented in Europe for the first time. NS further enabled detection of Anaplasma phagocytophilum (9.1 %), Coxiella burnetii (5.4 %) and Neoehrlichia mikurensis (1.8 %), as well as various endosymbionts of Rickettsia and Coxiella. Co-detection of multiple rickettsial and non-rickettsial bacteria were observed in 16.4 % of the pools with chromosome and plasmid-based contigs. In conclusion, non-targeted metagenomic sequencing was documented as a robust strategy capable of providing a broader view of the tick-borne bacterial pathogen spectrum., Competing Interests: Declaration of Competing Interest None, (Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.)