Purpose: The use of passaged chondrocytes is the current standard for autologous chondrocyte implantation (ACI). De-differentiation due to amplification and donor site morbidity are known drawbacks highlighting the need for alternative cell sources. Methods: Via clinically validated flow cytometry analysis, we compared the expression of human stem cell and cartilage markers (collagen type 2 (Col2), aggrecan (ACAN), CD44) of chondrocytes (CHDR), passaged chondrocytes for ACI (CellGenix™), bone marrow derived mesenchymal stem cells (BMSC), and synovial derived stem cells (SDSC). Results: Primary, human BMSC and SDSC revealed similar adipogenic, osteogenic, and chondrogenic differentiation potential and stem cell marker expression. However, the expression of the chondrogenic markers Col2 and ACAN was statistically significant higher in SDSC. CHDR and SDSC expressed ACAN and CD44 equally, but Col2 was expressed more strongly on the SDSC surface. The marker expression of SDSC from osteoarthritic joints (Kellgren-Lawrence score ≥3) versus normal knees (Kellgren-Lawrence score ≤2) did not differ. Similarly, there was no difference between temporarily frozen and fresh SDSC. Col2 and ACAN surface expression declined with further passaging, whereas CD44 remained unchanged. We observed the same effect after reducing the serum content. When comparing CHDR for ACI with SDSC of the same passage (P2/3), both Col2 and ACAN, correlating with clinical outcome, were expressed higher in SDSC. Conclusions: In summary, SDSC demonstrated high differentiation potential and a stable chondrogenic phenotype. They might therefore be better suitable for ACI than BMSC or passaged CHDR.