A total of 100 years have passed since the discovery of the protozoan Trypanosoma cruzi, the etiologic agent of Chagas' disease. Since its discovery, the molecular and cellular biology of this early divergent eukaryote, as well as its interactions with the mammalian and insect hosts, has progressed substantially. It is now clear that this parasite presents unique mechanisms controlling gene expression, DNA replication, cell cycle and differentiation, generating several morphological forms that are adapted to survive in different hosts. In recent years, the relationship between the chromatin structure and nuclear organization with the unusual transcription, splicing, DNA replication and DNA repair mechanisms have been investigated in T. cruzi. This article reviews the relevant aspects of these mechanisms in relation to chromatin and nuclear organization.