ABSTRACTProtozoan parasites of theLeishmania donovanicomplex are the causative agents of visceral leishmaniasis (VL), the most severe form of leishmaniasis, with high rates of mortality if left untreated.Leishmaniaparasites are transmitted to humans through the bite of infected female sandflies (Diptera:Phlebotominae), and approximately 500,000 new cases of VL are reported each year. In the absence of a safe human vaccine, chemotherapy, along with vector control, is the sole tool with which to fight the disease. Miltefosine (hexadecylphosphatidylcholine [HePC]), an antitumoral drug, is the only successful oral treatment for VL. In the current study, we describe the phenotypic traits ofL. donovaniclonal lines that have acquired resistance to HePC. We performed whole-genome and RNA sequencing of these resistant lines to provide an inclusive overview of the multifactorial acquisition of experimental HePC resistance, circumventing the challenge of identifying changes in membrane-bound proteins faced by proteomics. This analysis was complemented by assessment of thein vitroinfectivity of HePC-resistant parasites. Our work underscores the importance of complementary “omics” to acquire the most comprehensive insight for multifaceted processes, such as HePC resistance.