BackgroundBacterial epidemiology needs to understand the spread and dissemination of strains in a One Health context. This is important for highly pathogenic bacteria such asBacillus anthracis,Brucellaspecies,andFrancisella tularensis.Whole genome sequencing (WGS) has paved the way for genetic marker detection and high-resolution genotyping. While such tasks are established for Illumina short-read sequencing, Oxford Nanopore Technology (ONT) long-read sequencing has yet to be evaluated for such highly pathogenic bacteria with little genomic variations between strains. In this study, three independent sequencing runs were performed using Illumina, ONT flow cell version 9.4.1, and 10.4 for six strains of each ofBa.anthracis,Br. suisandF. tularensis.Data from ONT sequencing alone, Illumina sequencing alone and two hybrid assembly approaches were compared.ResultsAs previously shown, ONT produces ultra-long reads, while Illumina produces short reads with higher sequencing accuracy. Flow cell version 10.4 improved sequencing accuracy over version 9.4.1. The correct (sub-)species were inferred from all tested technologies, individually. Moreover, the sets of genetic markers for virulence, were almost identical for the respective species. The long reads of ONT allowed to assemble not only chromosomes of all species to near closure, but also virulence plasmids ofBa. anthracis. Assemblies based on nanopore data alone, Illumina data alone, and both hybrid assemblies correctly detected canonical (sub-)clades forBa. anthracisandF. tularensisas well as multilocus sequence types forBr. suis.ForF. tularensis,high-resolution genotyping using core-genome MLST (cgMLST) and core-genome Single-Nucleotide-Polymorphism (cgSNP) typing produced highly comparable results between data from Illumina and both ONT flow cell versions. ForBa. anthracis,only data from flow cell version 10.4 produced similar results to Illumina for both high-resolution typing methods. However, forBr. suis,high-resolution genotyping yielded larger differences comparing Illumina data to data from both ONT flow cell versions.ConclusionsIn summary, combining data from ONT and Illumina for high-resolution genotyping might be feasible forF. tularensisandBa. anthracis,but not yet forBr. suis.The ongoing improvement of nanopore technology and subsequent data analysis may facilitate high-resolution genotyping for all bacteria with highly stable genomes in future.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-023-09343-z.