The non-coding control region (NCCR) of polyomaviruses includes the promoters for early and late genes, a transcription enhancer and the origin of DNA replication. Particularly virulent variants of the human pathogens BKPyV and JCPyV, as well as of simian virus 40 (SV40), occur in vitro and in vivo. These strains often harbour rearrangements in their NCCR, typically deletions of some DNA segment(s) and/or duplications of others. Using an SV40-based model system we provide evidence that duplications of enhancer elements, whether from SV40 itself or from the related BKPyV and JCPyV, increase early gene transcription and replicative capacity. SV40 harbouring subsegments of the strong cytomegalovirus (HCMV) enhancer replicated better than the common ‘wild-type’ SV40 in the human cell lines HEK293 and U2OS. In conclusion, replacing the SV40 enhancer with heterologous enhancers can profoundly influence SV40’s infective capacity, underscoring the potential of small DNA viruses to overcome cell type and species barriers.