We hypothesized that altered fate of tendon-derived stem cells (TDSCs) might contribute to chondro-ossification and failed healing in the collagenase-induced (CI) tendon injury model. This study aimed to compare the yield, proliferative capacity, immunophenotypes, senescence, and differentiation potential of TDSCs isolated from healthy (HT) and CI tendons. TDSCs were isolated from CI and healthy Sprague-Dawley rat patellar tendons. The yield, proliferative capacity, immunophenotypes, and senescence of TDSCs (CI) and TDSCs (HT) were compared by colony-forming unit assay, BrdU assay, flow cytometry, and β-galactosidase activity assay, respectively. Their osteogenic and chondrogenic differentiation potentials and mRNA expression of tendon-related markers were compared using standard assays. More TDSCs, which showed a lower proliferative potential and a higher cellular senescence were present in the CI patellar tendons compared to HT tendons. There was a higher alkaline phosphatase activity and mineralization in TDSCs (CI) in both basal and osteogenic media. More chondrocyte-like cells and higher proteoglycan deposition, Sox9 and collagen type II expression were observed in TDSCs (CI) pellets upon chondrogenic induction. There was a higher protein expression of Sox9, but a lower mRNA expression of Col1a1, Scx, and Tnmd in TDSCs (CI) in a basal medium. In conclusion, TDSCs (CI) showed altered fate, a higher cellular senescence, but a lower proliferative capacity compared to TDSCs (HT), which might contribute to pathological chondro-ossification and failed tendon healing in this animal model.