A frequently overlooked category of genes in large-scale genome analyses is the nonprotein-coding genes. Specifically, the genomic organization of tRNA genes in Trypanosoma cruzi—a unicellular eukaryotic pathogen responsible for causing the debilitating human disease Chagas disease—has largely remained uncharted due to the previous incompleteness of the genome assembly. By utilizing advanced genomic techniques and improved assembly methods, the quality of genome assemblies has significantly improved, making these studies feasible. Here, we analyzed the genic content and distribution of tRNA genes in the nuclear genome of various strains compared with the genome organization of other related trypanosomatids. We found synteny in most of the tDNAs clusters between T. cruzi and Trypanosoma brucei, highlighting the significance of the genomic location of these Pol III-transcribed genes. A vast majority of the isoacceptor species are encoded by two genes, except for tDNASeC, which comprises a tandem of 11 copies in the core compartment associated with well-positioned nucleosomes. Finally, we describe a group of tRNA genes located at chromatin folding domain boundaries, potentially acting as chromatin insulators in T. cruzi.