ABSTRACTTicks in the familyIxodidaeare important vectors of zoonoses, including Lyme disease (LD), which is caused by spirochete bacteria from theBorreliella(Borrelia)burgdorferisensu latocomplex. The blacklegged tick (Ixodes scapularis) continues to expand across Canada, creating hot spots of elevated LD risk at the leading edge of its expanding range. Current efforts to understand the risk of pathogen transmission associated withI. scapularisin Canada focus primarily on targeted screens, while natural variation in the tick microbiome remains poorly understood. Using multiomics consisting of 16S metabarcoding and ribosome-depleted, whole-shotgun RNA transcriptome sequencing, we examined the microbial communities associated with adultI. scapularis(n= 32), sampled from four tissue types (whole tick, salivary glands, midgut, and viscera) and three geographical locations within a LD hot spot near Kingston, Ontario, Canada. The communities consisted of both endosymbiotic and known or potentially pathogenic microbes, including RNA viruses, bacteria, and aBabesiasp. intracellular parasite. We show that β-diversity is significantly higher between the bacterial communities of individual tick salivary glands and midguts than that of whole ticks. Linear discriminant analysis effect size (LEfSe) determined that the three potentially pathogenic bacteria detected by V4 16S rRNA sequencing also differed among dissected tissues only, including aBorreliastrain from theB. burgdorferi sensu latocomplex,Borrelia miyamotoi, andAnaplasma phagocytophilum. Importantly, we find coinfection ofI. scapularisby multiple microbes, in contrast to diagnostic protocols for LD, which typically focus on infection from a single pathogen of interest (B. burgdorferisensu stricto).IMPORTANCEAs a vector of human health concern, blacklegged ticks (Ixodes scapularis) transmit pathogens that cause tick-borne diseases (TBDs), including Lyme disease (LD). Several hot spots of elevated LD risk have emerged across Canada asI. scapularisexpands its range. Focusing on a hot spot in southeastern Ontario, we used high-throughput sequencing to characterize the microbiome of whole ticks and dissected salivary glands and midguts. Compared with whole ticks, salivary glands and midguts were more diverse and associated with distinct bacterial communities that are less dominated byRickettsiaendosymbiont bacteria and are enriched for pathogenic bacteria, including aB. burgdorferi sensu lato-associatedBorreliasp.,Borrelia miyamotoi, andAnaplasma phagocytophilum. We also found evidence of coinfection ofI. scapularisby multiple pathogens. Overall, our study highlights the challenges and opportunities associated with the surveillance of the microbiome ofI. scapularisfor pathogen detection using metabarcoding and metatranscriptome approaches.