Cassava mosaic disease is an enormous problem affecting the viability and productivity of cassava in all the developing regions in Africa. The disease is now known to be caused by a number of cassava-infecting begomoviruses. New viruses and viral strains continue to be discovered and due to the lack of cloning and sequencing facilities in many African laboratories, a simple, rapid and sensitive technique is needed for screening of cassava plantations. Here we report on the development of a heteroduplex mobility assay (HMA) which could be used for the testing of viral-infected cassava. The assay involves amplifying the highly conserved core region of the coat protein gene of field isolates followed by denaturing and annealing with a number of reference strains. The HMA profiles in this study were able to differentiate four different viral species and 11 different virus strains, and showed a good correlation with sequencing results and phylogenetic comparisons with other sequenced cassava viruses. This technique is sensitive and rapid and has the added advantage of being able to detect mixtures of viruses in field-grown cassava.