BACKGROUND: Trypanosomatid parasites are a group of protozoans that cause devastating diseases that disproportionately affect developing countries. These protozoans have developed several mechanisms for adaptation to survive in the mammalian host, such as extensive expansion of multigene families enrolled in host-parasite interaction, adaptation to invade and modulate host cells, and the presence of aneuploidy and polyploidy. Two mechanisms might result in "complex" isolates, with more than two haplotypes being present in a single sample: multiplicity of infections (MOI) and polyploidy. We have developed and validated a methodology to identify multiclonal infections and polyploidy using whole genome sequencing reads, based on fluctuations in allelic read depth in heterozygous positions, which can be easily implemented in experiments sequencing genomes from one sample to larger population surveys. RESULTS: The methodology estimates the complexity index (CI) of an isolate, and compares real samples with simulated clonal infections at individual and populational level, excluding regions with somy and gene copy number variation. It was primarily validated with simulated MOI and known polyploid isolates respectively from Leishmania and Trypanosoma cruzi. Then, the approach was used to assess the complexity of infection using genome wide SNP data from 497 trypanosomatid samples from four clades, L. donovani/L. infantum, L. braziliensis, T. cruzi and T. brucei providing an overview of multiclonal infection and polyploidy in these cultured parasites. We show that our method robustly detects complex infections in samples with at least 25x coverage, 100 heterozygous SNPs and where 5-10% of the reads correspond to the secondary clone. We find that relatively small proportions (≤ 7%) of cultured trypanosomatid isolates are complex. CONCLUSIONS: The method can accurately identify polyploid isolates, and can identify multiclonal infections in scenarios with sufficient genome read coverage. We pack our method in a single R script that requires only a standard variant call format (VCF) file to run ( https://github.com/jaumlrc/Complex-Infections ). Our analyses indicate that multiclonality and polyploidy do occur in all clades, but not very frequently in cultured trypanosomatids. We caution that our estimates are lower bounds due to the limitations of current laboratory and bioinformatic methods.