Giardia duodenalis is a widespread parasite of mammalian species, including humans. Due to its invariant morphology, investigations of aspects such as host specificity and transmission patterns require the direct genetic characterisation of parasites from faecal samples. We performed a sequence analysis of four genes (ssrRNA, β-giardin, glutamate dehydrogenase and triose phosphate isomerase) of 61 human isolates and 29 animal isolates. The results showed that multilocus genotypes (MLGs) can be readily defined for G. duodenalis isolates of assemblage A but not for assemblage B. Indeed, for assemblage A isolates, there was no evidence of intra-isolate sequence heterogeneity, and congruent genotyping results were obtained at the four genetic loci investigated. Sequence comparison and phylogenetic analysis showed that human-derived and animal-derived MLGs are different, and further indicated the presence of a new sub-assemblage (referred to as "AIII"), which was found exclusively in wild hoofed animals. On the other hand, there were variable levels of intra-isolate sequence heterogeneity (i.e., the presence of two overlapping nucleotide peaks at specific positions in the chromatograms, or "heterogeneous templates") in assemblage B isolates from humans and animals, and this prevented the unambiguous identification of MLGs. Furthermore, in five human isolates and one non-human primate isolate, the assignment to assemblage B was problematic, given that one of the four markers supported an assignment to assemblage A. These findings raise concerns about the interpretation of genotyping data based on single markers, and indicate the need to understand the mechanisms that are responsible for the differences between G. duodenalis assemblages A and B.