Recently, three novel human polyomaviruses were discovered: KIPyV, WUPyV and MCPyV. To study the genetic variability of these viruses, an evolutionary analysis of the large T antigen, small t antigen, VP1, VP2 and VP3 genes was carried out. Of the genes analyzed, only the small t antigen of KIPyV and MCPyV was found to be under statistically supported selective pressure. In KIPyV, 31 selected sites were found under positive selection (ω(2) = 1.54), with posterior probabilities above 0.75. Of these sites, 17 are located in the J domain and two of these are located in the HPDGG box. Thirteen of these sites were identified as being under positive selection at the 99% confidence level. Moreover, seven codons were under significant purifying selection, particularly in the J domain. In MCPyV, we confirmed the five sites already found to be under positive selection (posterior probabilities 0.99). Selective pressure analysis may provide useful information on viral evolution.