Generalist species, by definition, exhibit variation in niche attributes that promote survival in changing environments. Increasingly, phenotypes previously associated with a species, particularly those with wide or expanding ranges, are dissolving and compelling greater emphasis on population‐level characteristics. In the present study, we assessed spatial variation in diet characteristics, gut microbiome and associations between these two ecological traits across populations of coyotes Canis latrans. We highlight the influence of the carnivore community in shaping these relationships, as the coyote varied from being an apex predator to a subordinate, mesopredator across sampled populations. We implemented a scat survey across three distinct coyote populations in Michigan, USA. We used carbon (δ13C) and nitrogen (δ15N) isotopic values to reflect consumption patterns and trophic level, respectively. Corresponding samples were also paired with 16S rRNA sequencing to describe the microbial community and correlate with isotopic values. Although consumption patterns were comparable, we found spatial variation in trophic level among coyote populations. Specifically, δ15N was highest where coyotes were the apex predator and lowest where coyotes co‐occurred with grey wolves Canis lupus. The gut microbial community exhibited marked spatial variation across populations with the lowest operational taxonomic units diversity found where coyotes occurred at their lowest trophic level. Bacteriodes and Fusobacterium dominated the microbiome and were positively correlated across all populations. We found no correlation between δ13C and microbial community attributes. However, positive associations between δ15N and specific microbial genera increased as coyotes ascended trophic levels. Coyotes provide a model for exploring implications of niche plasticity because they are a highly adaptable, wide‐ranging omnivore. As coyotes continue to vary in trophic position and expand their geographic range, we might expect increased divergence within their microbial community, changes in physiology and alterations in behaviour.