Cell division is one of the most primordial cellular processes and is essential for life propagation. It is composed of crucial events, namely genome duplication, chromosomes segregation and membrane separation. Failure of these processes will have dramatic impacts on the development of any organisms, including cell death, polyploidy, or cell cycle arrest. In the case of bacterial pathogens, blocking cell division efficiently using targeted drugs is of interest to cure bacterial infections. However, despite its ancestral origin, the mechanisms of cell division have diverged amongst organisms. Given its crucial role, as well as the potential therapeutic applications, cell division is an intensive field of research and many aspects are yet to be fully understood. Amongst bacteria, the phyla Proteobacteria and Firmicutes have been intensively studied using their respective model organisms Escherichia coli and Bacillus subtilis. However, there is a paucity of research on cell division in Actinobacteria, the phylum that contains the world’s leading pathogen, Mycobacterium tuberculosis. In particular, the molecular players involved in the process of chromosome segregation and the timing of cell division have not been fully investigated. In the well-studied bacteria E. coli and B. subtilis the Min system determines the division site localization, while the nucleoid occlusion system (Noc or SlmA, respectively) determines the timing of division. In mycobacteria, however, no homologs for the Min or Noc systems have been identified thus far. However, the partitioning (par) genes are currently thought to be responsible for symmetric chromosome segregation. In addition, FtsK has been identified as a homolog of the B. subtilis protein SpoIIIE, which is responsible for rescuing DNA in the forming septum and pulling it in the forespore during starvation-induced sporulation. Overall, the role and dynamics of DNA, where FtsK might play a role, during mycobacterial cell division, still remain to be uncovered. Current methods to study DNA localization using snapshot microscopy typically utilise DNA dyes, such as DAPI, Hoechst, or SYTO. However, these compounds impair cell growth, preventing the dynamic study of mycobacterial division. To circumvent this shortcoming, I constructed a Mycobacterium smegmatis reporter strain expressing a Dendra2 fusion protein in-frame to the histone-like protein Hlp, and compared it to conventional DNA dyes. Additionally, I characterized FtsK and, for the first time, demonstrated its essentiality in M. smegmatis. As FtsK is essential I adapted a dual repression system to knock down FtsK, which proved to be a synthetic rescue with the par genes deletion. Using a custom-made microfluidic device and long-term time-lapse microscopy, the distribution and dynamics of both Hlp and FtsK fluorescent reporter proteins were followed at the single-cell level. This approach allowed the spatial and temporal characterization of these proteins with respect to other septal genes in wild-type cells as well as in Δpar mutants. Finally, I propose a new model where the par genes are important for chromosome orientation and FtsK is important for chromosome segregation. Altogether, this thesis represents a step forward towards a better understanding of the mechanisms underlying cell division in mycobacteria and in the long-term, a potential source of novel treatments to control the division of M. tuberculosis, for instance by targeting FtsK.