Phylogenetic analyses based on the major capsid protein sequence indicate that Merkel cell polyomavirus (MCPyV) and chimpanzee polyomaviruses (PtvPyV1, PtvPyV2), and similarly Trichodysplasia spinulosa-associated polyomavirus (TSPyV) and the orangutan polyomavirus (OraPyV1) are closely related. The existence of cross-reactivity between these polyomaviruses was therefore investigated. The findings indicated serological identity between the two chimpanzee polyomaviruses investigated and a high level of cross-reactivity with Merkel cell polyomavirus. In contrast, cross-reactivity was not observed between TSPyV and OraPyV1. Furthermore, specific antibodies to chimpanzee polyomaviruses were detected in chimpanzee sera by pre-incubation of sera with the different antigens, but not in human sera.