This paper presents the results of a long-term study to detect the presence of Puma concolor (Cougar) in eastern Canada. We installed 38 scratching posts to attract wild Cougars and collect hair samples in several national and provincial parks in Québec, New Brunswick, and Nova Scotia. A set of semi-nested primers was used to discriminate Cougar samples from other mammalian species based on variation in the 16S rRNA gene of the mitochondrial DNA. Our analyses performed on 476 hair samples revealed 19 positive identifications of Cougars in Québec and New Brunswick. Sequencing further showed that some specimens were from South America, whereas others had a North American origin. We discuss the implications of these results for the conservation of Cougars in eastern Canada.