Chronic wasting disease (CWD) is an efficiently transmitted spongiform encephalopathy of cervids. Whether CWD could represent a threat to non-cervid species remains speculative. Here we show that brain homogenates from several CWD-susceptible non-cervid species, such as ferrets and hamsters, support amplification of PrP^CWD by sPMCA, whereas brain homogenates from CWD-resistant species, such as laboratory mice and transgenic mice expressing human PrP^C [Tg(HuPrP) mice], do not. We also investigated whether several North American species that share the environment with cervids would support amplification of PrP^CWD by sPMCA. Three native rodent species, including voles and field mice, supported PrP^CWD amplification, whereas other species (e.g. prairie dog, coyote) did not. Analysis of PrP sequences suggests that an ability to support amplification of PrP^CWD in trans-species sPMCA is correlated with the presence of asparagine at position 170 of the substrate species PrP. Serial PMCA may offer insights into species barriers to transmission of CWD.