Djibouti is confronted with malaria resurgence, with malaria having been occurring in epidemic proportions since a decade ago. The current epidemiology of drug-resistant is not well known. Molecular markers were analyzed by targeted sequencing in 79 clinical isolates collected in Djibouti city in 2023 using the Miseq Illumina platform newly installed in the country. The objective of the study was to analyze the key codons in these molecular markers associated with antimalarial drug resistance. The prevalence of the mutant CVIET haplotype (92%) associated with chloroquine resistance and mutant haplotypes (7.4% SGEA and 53.5% IRN, respectively) associated with sulfadoxine-pyrimethamine resistance was high. By contrast, haplotypes associated with amodiaquine (YYY) or lumefantrine (NFD) resistance were not observed in any of the isolates. Although the "Asian-type" mutations associated with artemisinin resistance were not observed, the "African-type" PfK13 substitution, R622I, was found in a single isolate (1.4%) for the first time in Djibouti. Our genotyping data suggest that most Djiboutian isolates are resistant to chloroquine and sulfadoxine-pyrimethamine but are sensitive to amodiaquine, lumefantrine, and artemisinin. Nonetheless, the presence of an isolate with the R622I PfK13 substitution is a warning signal that calls for a regular surveillance of molecular markers of antimalarial drug resistance.
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