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Abstract Background Urine is routinely evaluated in dogs to assess health. Reference ranges for many urine properties are well established, but the scope of variation in these properties over time within healthy dogs is not well characterized. Objectives Longitudinally characterize urine properties in healthy dogs over 3 months. Animals Fourteen healthy client‐owned dogs. Methods In this prospective study, dogs were evaluated for health; then, mid‐stream free‐catch urine was collected from each dog at 12 timepoints over 3 months. Urine pH, urine specific gravity (USG), protein, cultures, and antimicrobial resistance profiles were assessed at each timepoint. Results Urine pH varied within and between dogs over time (Friedman's test: within P = .03; between P < .005). However, USG, protein, and bacterial diversity of urine were consistent within dogs over time, and only varied between dogs (Kruskal‐Wallis: between all P < .005). Antimicrobial resistant isolates were identified in 12 out of 14 dogs with 34 of 48 of the isolates demonstrating resistance to amoxicillin. Conclusions and Clinical Importance Urine pH should be assessed at multiple timepoints via pH meter before making clinical decisions. Mid‐stream free‐catch urine with high concentrations of bacteria (>105 CFU/mL) should not be considered the only indicator of urinary tract infection. Bacterial isolates from dogs in this study had widespread resistance to amoxicillin/oxacillin underscoring the need for antimicrobial stewardship.