Most canine microbiome trials are conducted in a controlled environment, with an unverified assumption that identical findings can be extrapolated to household dogs. In this study, the fecal microbiome of healthy beagles living in household or controlled environments was characterized with shotgun metagenomic sequencing and compared. Beagles were recruited from U.S. households (Pet, n = 54), a private kennel (Kennel, n = 39), and the University of Illinois at Urbana-Champaign (UIUC, n = 34). Dogs were fed an identical diet (fresh chicken, non-extruded and cooked) or diet type (kibble, various brands) for ≥ 7 days. Shannon diversity index was significantly higher in Kennel than Pet when fed both fresh and kibble, and significantly higher than UIUC when fed fresh only. β-Diversity was significantly different among the three cohorts on both diets. Mainly, species belonging to Actinobacteriota, Bacteroidota, Firmicutes_A, Firmicutes, and Proteobacteria were differentially abundant among the cohorts. Likewise, significant discriminant features in the functional data were observed among the cohorts after controlling for diet type. The findings demonstrate the living environment as an important factor in microbial composition, and further draw attention to possible limitations in the translation of findings between dogs in controlled versus free-living environments.