Canine distemper virus (CDV) is a highly contagious viral pathogen of worldwide distribution that can cause lethal disease in domestic dogs and other members of the family Canidae. Genetic diversity is found among reference strains and isolates of CDV, mainly in the haemagglutinin (H) protein, and this may be associated with the increasing incidence of distemper in dogs. CDV was isolated in Vero cells expressing canine signalling lymphocyte activation molecule (Vero.DogSLAM) from peripheral blood mononuclear cells and spleen of clinically diseased, previously vaccinated South African dogs. Direct fluorescence antibody test and electronmicroscopy were used to confirm the isolation procedure. Subsequently, RT-PCR was performed on the cell culture isolates, the amplified products were purified and the complete H gene was sequenced and phylogenetically analysed. The H gene of vaccines in use in South Africa was also sequenced and comparative analyses performed. However, the sequences obtained from the sick dogs showed 100% nucleotide identity and was different to that found in virus strains used in vaccines and in isolates reported from other parts of the world in GenBank. The results suggest that a novel CDV lineage may be present in South Africa and we conclude that a recent reversion of vaccine virus to virulence was not the cause of the clinical signs seen in dogs with a previous history of vaccination.