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Background: Giardia duodenalis is a leading cause of gastroenteritis worldwide. Humans are mainly infected by two different subtypes, i.e., assemblage A and B. Genotyping is hampered by allelic sequence heterozygosity (ASH) mainly in assemblage B, and by occurrence of mixed infections. Here we assessed the suitability of current genotyping protocols of G. duodenalis for epidemiological applications such as molecular tracing of transmission chains. Methodology/Principal findings: Two G. duodenalis isolate collections, from an outpatient tropical medicine clinic and from several primary care laboratories, were characterized by assemblage-specific qPCR (TIF, CATH gene loci) and a common multi locus sequence typing (MLST; TPI, BG, GDH gene loci). Assemblage A isolates were further typed at additional loci (HCMP22547, CID1, RHP26, HCMP6372, DIS3, NEK15411). Of 175/202 (86.6%) patients the G. duodenalis assemblage could be identified: Assemblages A 25/175 (14.3%), B 115/175 (65.7%) and A+B mixed 35/175 (20.0%). By incorporating allelic sequence heterozygosity in the analysis, the three marker MLST correctly identified 6/ 9 (66,7%) and 4/5 (80.0%) consecutive samples from chronic assemblage B infections in the two collections, respectively, and identified a cluster of five independent patients carrying assemblage B parasites of identical MLST type. Extended MLST for assemblage A altogether identified 5/6 (83,3%) consecutive samples from chronic assemblage A infections and 15 novel genotypes. Based on the observed A+B mixed infections it is estimated that only 75% and 50% of assemblage A or B only cases represent single strain infections, respectively. We demonstrate that typing results are consistent with this prediction. Conclusions/Significance: Typing of assemblage A and B isolates with resolution for epidemiological applications is possible but requires separate genotyping protocols. The high frequency of multiple infections and their impact on typing results are findings with immediate consequences for result interpretation in this field. Author summary: Giardia duodenalis is a leading cause of gastroenteritis worldwide. Humans are mainly infected by the two different genetic subtypes, assemblage A and B. Molecular typing tools for epidemiological applications such as tracking transmission, attribution to a source and outbreak investigations have been developed and are highly desirable. However, to what degree the tetraploid genome with allelic sequence heterogeneity (ASH), and the frequent occurrence of mixed, assemblage A and B infections hamper performance is unclear. Here, we assessed the suitability of current genotyping protocols for deciphering the molecular epidemiology of G. duodenalis. Against a common reporting bias, we incorporated ASH in the analysis and we show that typing with resolution for epidemiological applications is possible for both, assemblage A and B isolates, but requires separate protocols. We also demonstrate how the high frequency of multiple infections overall impacts on typing results, which has immediate consequences for result interpretation in this field. [ABSTRACT FROM AUTHOR]