Sequence analysis ofPlasmodium falciparumparasites is informative in ensuring sustained success of malaria control programmes. Whole-genome sequencing technologies provide insights into the epidemiology and genome-wide variation ofP. falciparumpopulations and can characterise geographical as well as temporal changes. This is particularly important to monitor the emergence and spread of drug resistantP. falciparumparasites which is threatening malaria control programmes world-wide. Here, we provide a detailed characterisation of genome-wide genetic variation and drug resistance profiles in asymptomatic individuals in South-Western Mali, where malaria transmission is intense and seasonal, and case numbers have recently increased. Samples collected from Ouélessébougou, Mali (2019–2020; n = 87) were sequenced and placed in the context of older Malian (2007–2017; n = 876) and African-wide (n = 711)P. falciparumisolates. Our analysis revealed high multiclonality and low relatedness between isolates, in addition to increased frequencies of molecular markers for sulfadoxine-pyrimethamine and lumefantrine resistance, compared to older Malian isolates. Furthermore, 21 genes under selective pressure were identified, including a transmission-blocking vaccine candidate (pfCelTOS) and an erythrocyte invasion locus (pfdblmsp2). Overall, our work provides the most recent assessment ofP. falciparumgenetic diversity in Mali, a country with the second highest burden of malaria in West Africa, thereby informing malaria control activities.