Background: Plasmodium malariae is a globally distributed and often neglected malaria parasite thatoccurs as a chronic, low-density, mixed-species infections in humans. Its genetic diversityhas so far only been assessed using conventional length and microsatellite genotypingmethods. These methods typically have low sensitivity to detect minority parasite clonescompared to sequencing-based methods. In this study, amplicon deep sequencing (AmpSeq)was applied to 4 polymorphic genotyping markers (pmama1 FP, pmama1 SP, pmmsp1 F3and 18S rRNA) using 96 P. malariae infected samples from Gabon. One sample wasassessed across 12 timepoints to evaluate marker performance. AmpSeq reported greaterhaplotype diversity, Multiplicity of Infection (MOI) and expected Heterozygosity (He) thanpreviously reported by conventional methods. The number of unique haplotypes for themarkers ranged from 9 to 26, the average MOI from 2 to 3.77 and He ranged from 0.81 to0.93. The combined use of pmama1 FP, pmmsp1 F3 and 18S rRNA produced consistentdiversity estimates and also performed reliably in the longitudinal study set. More than half ofthe sequences detected had not been previously documented in global databases, indicatingnotable variation in Gabon.