In the context of global bacterial pathogen surveillance, it is crucial to ensure interoperability and harmonized data. Several systems are currently implemented for such surveillance, being designed to compare bacteria and identify outbreak clusters based on core genome MultiLocus Sequence Typing (cgMLST) profiles. Among the different approaches available to generate bacterial cgMLST profiles, our research used an assembly-based approach - as implemented in the chewBBACA tool - according to European Food Safety Authority (EFSA) guidelines. Simulations of short-read sequencing were conducted for 27 bacterial pathogen species of interest in animal, plant, and human health to evaluate the repeatability and reproducibility of cgMLST profiles. Various quality parameters, such as read quality and depth of sequencing were applied, and several read simulations and genome assemblies were repeated using three commonly used tools: SPAdes, Unicycler and Shovill. The results highlighted bioinformatic variability in cgMLST profiles, which appears unrelated to the assembly tools, but rather induced by the intrinsic composition of the genomes themselves. This variability observed in simulated sequencing was further validated with real data for five of the bacterial pathogens studied.