Mesenchymal stem cells (MSCs) constitute a great promise for regenerative therapy, but these cells are difficultly recovered in large amounts. A potent alternative is the stromal vascular fraction (SVF), non-cultured MSCs, separated from adipose tissue (AT). We aim to evaluate AT harvesting site effect on the SVF cells’ quantity and quality in dogs. Subcutaneous abdominal fat, falciform ligament and peri-ovarian fat were sampled. After SVF isolation, the trypan blue exclusion test and a hemocytometer were used to assess the cell viability and cellular yield. SVF cells were labeled for four surface antigenic markers, clusters of differentiation CD90, CD44, CD29, and CD45, and then examined by flow cytometry. Semi-quantitative RT-PCR was used to evaluate the gene expression of the former markers in addition to OCT-4 and CD34. SVF cells in the peri-ovarian AT recorded the highest viability% (99.63 ± 0.2%), as well as a significantly higher cellular yield (36.87 ± 19.6 × 106 viable cells/gm fat, p < 0.001) and a higher expression of adipose-derived mesenchymal stem cells AD-MSCs surface markers than that of other sites. SVF cells from the peri-ovarian site revealed a higher expression of MSC markers (CD90, CD44, and CD29) and OCT-4 compared to the other sites, with weak CD45 and CD34 expressions. The positive OCT-4 expression demonstrated the pluripotency of SVF cells isolated from different sites. To conclude, the harvesting site is a strong determinant of SVF cells’ quantity and quality, and the peri-ovarian site could be the best AT sampling site in dogs.