Background/Aim: Olfactory ensheathing cells (OECs) are widely studied for neural repair, yet OB- and OM-derived primary cultures differ in accessibility and cellular composition. This study aimed to establish donor-matched canine OB- and OM-derived primary cultures using harmonized isolation conditions and to quantify source-dependent differences in culture composition and proliferative activity. Materials and Methods: Olfactory bulbs (OBs) and olfactory mucosa (OM) were collected post-mortem from client-owned dogs (n = 10). Primary cultures were established under identical enzymatic dissociation and culture conditions. Culture composition was quantified by immunocytochemistry using p75^NTR (OEC marker) and fibronectin (fibroblast-associated marker), with an epithelial fraction assessed morphologically in OM. Proliferation was assessed by Ki-67 labeling using the Muse^® Ki-67 kit (n = 5 donors/group). Results: Both tissues yielded viable primary cultures. OB-derived cultures had a higher OEC fraction than OM-derived cultures (60.7 ± 6.4% vs. 39.0 ± 6.2%), whereas OM cultures consistently included an epithelial component (27.0 ± 6.6%). Ki-67 labeling was higher in OB-derived cultures than OM-derived cultures (30.2 ± 6.2% vs. 13.0 ± 2.5%; Welch’s t-test p = 0.0018). Conclusions: Canine OB and OM generate source-distinct primary cultures under standardized conditions: OB-derived cultures are OEC-enriched and more proliferative in vitro, while OM-derived cultures are more heterogeneous. These findings inform future optimization of OM-based protocols and motivate functional assays to test regenerative efficacy.