The prevalence of Giardia duodenalis was determinate in faecal samples from dogs and cats in Madrid, Spain and molecular characterisation of isolates. A total of 604 and 144 faecal samples from dogs and cats, respectively, were analysed by routine coprological methods. The prevalence of G. duodenalis was 16.4 % (99/604) in dogs and 4.2 % (6/144) in cats. Sixty-four G. duodenalis isolates (63 from dogs and 1 from a cat) were characterised using glutamate dehydrogenase and β-giardin genes by PCR-RFLP. The single cat sample showed a mixed infection by assemblages A + F. The assemblages found in the dog samples were A, B, C, D and E, both as single and as mixed infections. The zoonotic assemblages A and B were found in 56 (88.8 %) G. duodenalis-positive samples with 15.9 % of samples having assemblage A (10/63) and 73 % of samples with assemblage B (46/63), indicating high potential zoonotic risk and public health significance.