The kinetochore is a highly complex proteinaceous structure located at the primary constriction of mitotic chromosomes. Here, it performs an essential role in accurate chromosome segregation. Recently, much interest has been directed towards the Constitutive Centromere Associated Network (CCAN) components, as they participate in the formation of a scaffold involved in kinetochore assembly. It is therefore important to fully understand their role, and their distribution, at the kinetochore. Although many kinetochore proteins have already been identified, it is still unclear how centromeric chromatin folds to form the structure of the inner kinetochore. This is an interesting yet still open field of study, where the literature reports are still quite divided. In this study we take advantage of the high homologous recombination efficiency in DT40 B-lymphoma chicken cell lines, allowing the generation of conditional knockouts and deletion cell lines of several centromere proteins, subsequently engineered to stably express GFP:CENP-A. In the parental cell line the unfolding properties of the centromeric region were investigated by using TEEN buffer. Using fluorescence microscopy we were able to measure the length of many unfolded centromeric chromatin fibres, from both interphase and mitotic samples, based on the signal of GFP:CENP-A. A multi-peak analysis revealed the presence of discrete populations of fibres, recognised as peaks, in both interphase and mitotic samples. Compared with interphase, mitotic centromeres showed a greater level of compaction. Next, mutants for CCAN components, blocked in mitosis, were subjected to centromere chromatin unfolding. Results revealed that mitotic kinetochores depleted of CENP-C and CENP-S behaved similarly to the parental interphase samples, suggesting a role of those proteins in maintaining kinetochore structure. In contrast, CENP-O, CENP-H and CENP-I depletion did not seem to weaken the structure of the kinetochore. Additionally, we tested a hypothesis revealed by the multi-peak analyses, that chromatin layers exist in the inner kinetochore. Our data, when combined with published electron microscopy and crystallography measurements of centromere/kinetochore components, allowed us to assemble a robust and mathematically viable model that supports a boustrophedon organisation of the kinetochore chromatin. Finally, characterization studies of the novel kinetochore protein CENP-Z were performed. An involvement of CENP-Z in controlling the levels of di-methylation on lysine 4 of histone H3 was shown. This work represents an advance in our understanding of kinetochore structure in vertebrates.