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Abstract Background Accurate variant calls from whole genome sequencing (WGS) of Plasmodium falciparum infections are crucial in malaria population genomics. Here a falciparum variant calling pipeline based on GATK version 4 (GATK4) was optimized and applied to 6626 public Illumina WGS samples. Methods Control WGS and accurate PacBio assemblies of 10 laboratory strains were leveraged to optimize parameters that control the heterozygosity, local assembly region size, ploidy, mapping and base quality in both GATK HaplotypeCaller and GenotypeGVCFs. From these controls, a high-quality training dataset was generated to recalibrate the raw variant data. Results On current high-quality samples (read length = 250 bp, insert size = 405–524 bp), the optimized pipeline shows improved sensitivity (86.6 ± 1.7% for SNPs and 82.2 ± 5.9% for indels) compared to the default GATK4 pipeline (77.7 ± 1.3% for SNPs; and 73.1 ± 5.1% for indels, adjusted P