Background: Gastrointestinal cancer accounts for approximately 8% of all canine malignancies. Early detection of cancer may have a tremendous impact on both treatment options and prognosis. MicroRNAs (miRNAs), a class of noncoding RNAs that can be found stably expressed in body fluids and feces, have been suggested as valuable human cancer biomarkers. Objectives: The purpose of the study was to investigate the feasibility of detecting miRNAs in canine feces and to determine the miRNA stability in fecal samples stored at different temperatures for different duration. Methods: The levels of 4 Canine familiaris (cfa) miRNAs (cfa-miR-16, cfa-miR-20a, cfa-miR-21, and cfa-miR-92a) were investigated by quantitative real-time PCR(qPCR) in fecal samples from 10 healthy dogs. Fecal samples were collected at 3 different time points and samples from the first time point were stored at different temperatures and for a different duration. Results: A statistically significant difference was found in miRNA levels from samples stored at room temperature compared with samples stored at −20°C for cfa-miR-16 and cfa-miR-21. No significant difference was found in the level of the investigated miRNAs over time. Conclusions: Overall, miRNAs are present in dog feces at measurable levels. Some miRNAs seem to be subject to a higher degree of degradation in samples stored at room temperature for 24 hours compared with samples frozen after collection at −20°C. The investigated miRNAs were stably expressed over time. This study provides the basis for further research on miRNA expression profiles as biomarkers for gastrointestinal cancer in dogs.