ABSTRACT Genetic linkage using recombinant progeny from controlled experimental crosses is a powerful tool for locating the genetic determinants that control important phenotypes. We have perfected the methods for quantitative trait loci (QTL) mapping of malaria parasite traits using the humanized mouse model and have optimized bulk segregant analysis (BSA) for streamlined identification of loci and prioritization of genes in the locus for functional validation. The recent emergence of artemisinin resistant (ART-R) parasites in East Africa hastens the need to understand the genes and mechanisms of fit, drug resistant parasites. The looming crisis of spreading resistance to ART combination therapies (ACT) across Africa hinges on unique features associated with different kelch13 mutations appearing in different locales with different propensities to expand in frequency. Current surveillance is to monitor kelch13 mutants and to assess cumbersome clinical phenotypes such as patient clearance half-life and the labor-intensive Ring Stage Survival assay (RSA). We will partner with African researchers to target this emerging crisis by generating experimental crosses between parasite strains that differ for important ART-R and ACT-related traits to map the location of the genes and their mutations that control these phenotypes. We will use both BSA selections (Aim 1) and clone-based QTL mapping (Aim 2). By judicious choice of parental lines for constructing targeted experimental crosses Aim 1 will combine BSA with refined selection schemes; we hypothesize that we can rapidly characterize kelch13 mutations and their phenotypic impacts, as well as their broader whole genome profile to inform and improve surveillance. In Aim 2, we will deploy a range of novel phenotypes we developed in the initial funding period, including a high-throughput adaptation of the traditional RSA, along with an expansion of dosing regimens and recovery times. Aim 3 will apply these approaches to individual progeny growth rates and comprehensive pair-wise competitive fitness. Working in concert with Core B, we will identify additional genes and mechanisms that contribute to fit forms of ART-R. This approach will allow us to test hypotheses about resistance evolution and characterize partner genes and pathways affected by drug selections and identify the factors that influence the origins and spread of resistance.