The numbers of patients with tuberculosis (TB) caused by resistant strains are still alarming. Therefore, it is necessary to determine resistance more quickly and precisely, than it is with the currently used phenotypic and genotypic methods. In recent years, technological advances have been made and the whole-genome sequencing (WGS) method has been introduced as a part of routine diagnostics in clinical laboratories. Comparing a wide range of mycobacterial genomic variations with a reference genome leads to a consistent evaluation of molecular-epidemiology and resistance of Mycobacterium tuberculosis (M. tuberculosis) to a wide range of anti-TB drugs. The quality of the obtained sequencing data is closely related to the type of sample and the method used for DNA extraction and sequencing library preparation. Moreover, the correct interpretation of results is also influenced by a bioinformatic data processing. A large number of bioinformatics pipelines are currently available, the sensitivity of which varies due to the different sizes of databases containing relevant mutations. This review focuses on the individual steps included in the sequencing workflow and factors that may affect the interpretation of final results.