ABSTRACT Genetic crosses coupled with linkage mapping have provided an outstandingly successful approach for locating the genetic determinants of biomedically important traits such as drug resistance and host specificity in P. falciparum malaria. In the initial funding period of this Program Project (P01), we made great strides in transitioning Plasmodium crosses from the original model using chimpanzees to the human-liver chimeric mouse infused with human red blood cells (the FRG huHep/huRBC mouse) to generate more than 30 crosses in a span of 5 years. This included many replicate crosses of 6 different parental combinations that produced nearly a thousand new cloned progeny. This capacity allowed for optimizations that drove our development of the bulk segregant analysis (BSA) methodology and put within reach the ability to use experimental crosses of new clinical isolates to test specific hypotheses about emerging drug resistance. The combination of routine and replicated experimental crosses with BSA has shifted the challenge from making crosses and identifying genetic loci to instead prioritizing the rapid identification of genes and mechanisms underling parasite drug resistance and fitness. Our capstone discovery identified the role of pfaat1 as an epistatic partner with pfcrt in the evolution of chloroquine resistance that influences the balance between resistance and compensatory fitness, a crucial determinant of how new drug resistances emerge and spread. The overall goal of this P01 renewal proposal is to continue to advance this technology to track in real-time the alarming emergence of artemisinin resistance (ART-R) and its partner drugs used in artemisinin combination therapies (ACTs) in East Africa, an imminent threat to reverse decades of progress against morbidity and mortality due to malaria on the continent where more than 90% of malaria deaths occur. Coding mutations in the kelch13 gene that are strongly associated with ART-R and serve as the only markers for surveillance. However, mutations in kelch13 generate a wide range of resistance levels and fitness effects, and how these effects are compensated by other structural or regulatory changes in the genome remains unknown. Relying on 3 Research Projects, supported by 2 Scientific Cores, we will use targeted experimental genetic crosses to (i) dissect the genetic complexity of ART-R, (ii) clarify the role of kelch13, (iii) define the regulators and partner genes that control ART-R and emerging resistance to lumefantrine, the predominant ACT partner drug in Africa and (iv) establish a sustainable and user-centric valuable community resource including our methodological pipeline, data and biological materials. In the process, we will expand our BSA drug selection protocols to include cutting edge single cell RNAseq as the next step in building this powerful community resource for real-time solutions to clinically urgent questions.