Chronic wasting disease (CWD) was first identified in Wisconsin (USA) in white-tailed deer (Odocoileus virginianus) in February 2002. To determine if prion protein gene (Prnp) allelic variability was associated with CWD in white-tailed deer from Wisconsin, we sequenced Prnp from 26 CWD-positive and 100 CWD-negative deer. Sequence analysis of Prnp suggests that at least 86–96% of the white-tailed deer in this region have Prnp allelic combinations that will support CWD infection. Four Prnp alleles were identified in the deer population, one of which, resulting in a glutamine to histidine change at codon 95, has not been previously reported. The predominant allele in the population encodes for glutamine at codon 95, glycine at codon 96, and serine at codon 138 (QGS). Less abundant alleles encoded QSS, QGN, and HGS at the three variable positions. Comparison of CWD-positive with CWD-negative deer suggested a trend towards an over-representation of the QGS allele and an under-representation of the QSS allele.