This study aimed to compare clonogenicity, proliferation, stem cell-related marker expression, senescence, and differentiation potential of rat patellar tendon-derived stem cells (TDSCs) at early (P5), mid (P10), and late (P20, P30) passages. The clonogenicity of the cells was assessed by colony-forming assay and their proliferative potential was assessed by bromodeoxyuridine assay. The surface expression of CD90 and CD73 was assessed by flow cytometry. The cellular senescence was assessed by β-galactosidase activity. The adipogenic, chondrogenic, and osteogenic differentiation potentials of TDSCs were assessed by standard assays after induction. The mRNA expression of tendon-related markers, scleraxis (Scx) and tenomodulin (Tnmd), was measured by quantitative real-time reverse transcription-polymerase chain reaction. Both the colony numbers and proliferative potential of TDSCs increased with passaging. Concomitantly, there was significant upregulation of β-galactosidase activity with TDSC passaging. The subculture of TDSCs downregulated the expression of CD90 and CD73. Lipid droplets were formed in the early and mid passages of TDSCs upon adipogenic induction, but were absent in the late passages. The expression of peroxisome proliferator activator receptor gamma 2 (PPARγ2) and CCAAT/enhancer binding protein alpha (C/EBPα) in TDSCs after adipogenic induction decreased with passaging. Chondrogenesis, proteoglycan deposition, collagen type II protein expression, collagen type 2A1 (Col2AI), and aggrecan (Acan) mRNA expression were less in pellets formed with later passages of TDSCs after chondrogenic induction. The expression of Scx and Tnmd was lower in the late, compared with early and mid, passages of TDSCs. However, matrix mineralization and expression of alkaline phosphatase (Alpl) and osteocalcin (Bglap) mRNA after osteogenic induction increased with TDSC passaging. Researchers and clinicians should consider the changes of stem cell-related properties of TDSCs when multiplying them in vitro for tissue engineering.