Chagas disease is the most important parasitic disease in the Western Hemisphere, caused by the protozoan parasite Trypanosoma cruzi. Understanding and controlling the disease has been challenging due to the extensive complexity of transmission cycles, which involve a wide variety of vectors and host species, highly diverse parasite strains, and clinical presentations. In recent years, the use of next-generation sequencing (NGS) has allowed several breakthroughs to be made in addressing some of these challenges that we review here. Sequencing of parasite genomes and transcriptomic studies have shed light on parasite diversity, its differentiation and responses to drug treatment. T. cruzi has the ability to infect any mammalian species, and the elucidation of parasite–host interactions has been enhanced in great part by NGS technologies. Thus, host and parasite genetic diversity has been shown to play a role in Chagas disease pathogenesis, and the host microbiome is also likely involved in these interactions. Finally, NGS has provided insight into the molecular ecology of triatomine vectors and parasite transmission. Bug species, blood feeding sources, T. cruzi parasite diversity, and triatomine gut microbiota all play a role in transmission cycles and risk of infection. Characterizing these interactions can lead to improved control. While these studies provide a robust basis to describe the complexity of Chagas disease, their future integration is poised to provide further insight into disease biology and ecology, and lead to new tools and approaches to disease control.