Microbiome studies generally focus on the gut microbiome, which is composed of a large proportion of commensal bacteria. Here we propose a first analysis of the liver microbiome using next generation sequencing as a tool to detect potentially pathogenic strains. We used Peromyscus leucopus, the main reservoir host species of Lyme disease in eastern North America, as a model and sequenced V5-V6 regions of the 16S gene from 18 populations in southern Quebec (Canada). The Lactobacillus genus was found to dominate the liver microbiome. We also detected a large proportion of individuals infected by Bartonella vinsonii arupensis, a human pathogenic bacteria responsible for endocarditis, as well as Borrelia burgdorferi, the pathogen responsible for Lyme disease in North America. We then compared the microbiomes among two P. leucopus genetic clusters occurring on either side of the St. Lawrence River, and did not detect any effect of the host genotype on their liver microbiome assemblage. Finally, we report, for the first time, the presence of B. burgdorferi in a small mammal host from the northern side of the St. Lawrence River, in support of models that have predicted the northern spread of Lyme disease in Canada.