Tendon stem/progenitor cells (TSPCs) are a potential cell source for tendon tissue engineering. The striking morphological and structural changes of tendon tissue during development indicate the complexity of TSPCs at different stages. This study aims to characterize and compare post-natal rat Achilles tendon tissue and TSPCs at different stages of development. The tendon tissue showed distinct differences during development: the tissue structure became denser and more regular, the nuclei became spindle-shaped and the cell number decreased with time. TSPCs derived from 7 day Achilles tendon tissue showed the highest self-renewal ability, cell proliferation, and differentiation potential towards mesenchymal lineage, compared to TSPCs derived from 1 day and 56 day tissue. Microarray data showed up-regulation of several groups of genes in TSPCs derived from 7 day Achilles tendon tissue, which may account for the unique cell characteristics during this specific stage of development. Our results indicate that TSPCs derived from 7 day Achilles tendon tissue is a superior cell source as compared to TSPCs derived from 1 day and 56 day tissue, demonstrating the importance of choosing a suitable stem cell source for effective tendon tissue engineering and regeneration.