Recently, polyomaviruses KI and WU were identified in the airways of patients with acute respiratory symptoms. The epidemiology and pathogenesis of these two viruses are not fully understood, and the development of molecular assays, such as Real Time PCR, was useful for examining their biology and role in different clinical syndromes. The evaluation of different target regions for the amplification of polyomaviruses KI and WU, comparing published primer/probe sets and sets designed in the laboratory is described and was used for testing 175 clinical specimens (84 stools and 91 tonsils). The results showed that the laboratory designs were more sensitive for the detection of polyomaviruses KI and WU DNA in clinical samples. The choice of the primer/probe set, and primarily of the region for amplification, may be relevant for understanding the pathogenic role of viruses such as polyomaviruses KI and WU.