Trypanosomes are eukaryotic, unicellular parasites, such as Trypanosoma brucei, which causes sleeping sickness, and Trypanosoma cruzi, which causes Chagas disease. Genomes of these parasites comprise core regions and species-specific disruptive regions that encode multigene families of surface glycoproteins. Few transcriptional regulators have been identified in these parasites, and the role of spatial organization of the genome in gene expression is unclear. Here we mapped genome-wide chromatin interactions in T. cruzi using chromosome conformation capture (Hi-C), and we show that the core and disruptive regions form three-dimensional chromatin compartments named C and D. These chromatin compartments differ in levels of DNA methylation, nucleosome positioning and chromatin interactions, affecting genome expression dynamics. Our data reveal that the trypanosome genome is organized into chromatin-folding domains and transcription is affected by the local chromatin structure. We propose a model in which epigenetic mechanisms affect gene expression in trypanosomes.