ABSTRACTAtovaquone-proguanil (AP) is used as treatment for uncomplicated malaria, and as a chemoprophylactic agent againstPlasmodium falciparum. Imported malaria remains one of the top causes of fever in Canadian returning travelers. Twelve sequential whole-blood samples before and after AP treatment failure were obtained from a patient diagnosed withP. falciparummalaria upon their return from Uganda and Sudan. Ultradeep sequencing was performed on thecytb, dhfr,anddhpsmarkers of treatment resistance before and during the episode of recrudescence. Haplotyping profiles were generated using three different approaches:msp2-3D7agarose and capillary electrophoresis, andcpmpusing amplicon deep sequencing (ADS). A complexity of infection (COI) analysis was conducted.De novo cytbY268C mutants strains were observed during an episode of recrudescence 17 days and 16 h after the initial malaria diagnosis and AP treatment initiation. No Y268C mutant reads were observed in any of the samples prior to the recrudescence. SNPs in thedhfranddhpsgenes were observed upon initial presentation. The haplotyping profiles suggest multiple clones mutating under AP selection pressure (COI > 3). Significant differences in COI were observed by capillary electrophoresis and ADS compared to the agarose gel results. ADS usingcpmprevealed the lowest haplotype variation across the longitudinal analysis. Our findings highlight the value of ultra-deep sequencing methods in the understanding ofP. falciparumhaplotype infection dynamics. Longitudinal samples should be analyzed in genotyping studies to increase the analytical sensitivity.