Neglected tropical diseases (NTDs) mainly affect poor people living in rural areas of tropical and subtropical regions of the world. The study of gene function in the pathogens that cause these diseases has been delayed by inadequate genome editing techniques, lack of the interference system, genomic plasticity, or specific genomic features that hinder these processes. CRISPR-Cas offers a precise molecular application for targeted genome modification with various potential applications. Thus, CRISPR-Cas technology has opened new avenues for genome-wide studies in NTDs, providing insights into the biology of these parasites and enabling significant advances in genome editing. In this chapter, we show the relevance of CRISPR-Cas for studying agents causing tropical diseases, with emphasis mainly on kinetoplastids and apicomplexans. We will discuss how CRISPR-Cas has been used to edit genomes, investigate the molecular interactions between parasites and their hosts, develop high-throughput screening assays for identifying compounds with antiparasitic activity, or detect specific sequences for developing rapid and accurate diagnostic tests for parasitic infections. Additionally, this methodology has opened the possibility of performing genomic scale studies, which not only allow the study and functional characterization of genes but also open the possibility of searching for new treatments using their molecular targets and mechanisms, a challenge of great importance, considering that current treatments for all these diseases are inefficient and have the problem of drug resistance. Finally, we discuss how the CRISPR-Cas system could be used to study the gene expression regulation and epigenetic modifications involved in genomic functions.