A PCR-RFLP genotyping tool was developed and used to characterise morphologically identical isolates of Giardia duodenalis from a variety of host species. Primers were designed to amplify a 432bp region of the glutamate dehydrogenase gene (gdh) from genetic Assemblages AI, AII, BIII, BIV, C, D and E of G. duodenalis. DNA extracted from cultured Giardia trophozoites, Giardia cysts purified from faeces and directly from whole faeces was amplified and sequenced at the gdh and 18SrDNA loci. The gdh sequences were identical with published gdh sequences for each assemblage with a few exceptions. However, in some cases genotyping results obtained using gdh differed from 18SrDNA genotyping results. From gdh sequence information a PCR-RFLP profile was identified for each of the genetic assemblages. PCR-RFLP is a reproducible, reliable and sensitive method for genotyping Giardia. Eight human, 12 cat, 9 dog and 16 cattle faecal isolates were genotyped using PCR-RFLP. This method allows G. duodenalis isolates from human-beings, their companion animals and livestock to be genotyped directly from faeces, leading to valuable information about Giardia genotypes in population without the need for in vitro/in vivo amplification.