Multiplicity of infection (MOI), also termed complexity of infection (COI), is defined as the number of genetically distinct parasite strains co-infecting a single host, which is an important indicator of malaria epidemiology. PCR-based genotyping often underestimates MOI. Next generation sequencing technologies provide much more accurate and genome-wide characterization of polyclonal infections. However, complete haplotype characterization of multiclonal infections remains a challenge due to PCR artifacts and sequencing errors, and requires efficient computational tools. In this review, the advantages and limitations of current molecular approaches to determine multiplicity of malaria parasite infection are discussed.