In order to improve lithium ion batteries it is important to characterise real electrode geometries and understand how their 3D structure may affect performance. In this study, high resolution synchrotron nano-CT was used to acquire 3D tomography datasets of mesocarbon microbead (MCMB) based anodes down to a 16 nm voxel size. A specimen labelling methodology was used to produce anodes that enhance the achievable image contrast, and image processing routines were utilised to successfully segment features of interest from a challenging dataset. The 3D MCMB based anode structure was analysed revealing a heterogeneous and bi-modally distributed microstructure. The microstructure was quantified through calculations of surface area, volume, connectivity and tortuosity factors. In doing so, two different methods, random walk and diffusion based, were used to determine tortuosity factors of both MCMB and pore/electrolyte microstructures. The tortuosity factors (2–7) confirmed the heterogeneity of the anode microstructure for this field of view and demonstrated small MCMB particles interspersed between large MCMB particles cause an increase in tortuosity factors. The anode microstructure was highly connected, which was also caused by the presence of small MCMB particles. The complexity in microstructure suggests inhomogeneous local lithium ion distribution would occur within the anode during operation.