Skeletal stem cells (SSCs) are a population of progenitor cells which give rise to postnatal skeletal tissues including bone, cartilage and bone marrow stroma, however not to adipose, haematopoietic or muscle tissue. Growth plate chondrocytes exhibit the ability of continuous proliferation and differentiation, which contributes to the continuous physiological growth. The growth plate has been hypothesized to contain SSCs which exhibit a desirable differentiation capacity to generate bone and cartilage. Due to the heterogeneity of the growth plate chondrocytes, SSCs in the growth plate are not well studied. The present study used cluster of differentiation (CD) 146 and CD105 as markers to isolate purified SSCs. CD105(+) SSCs and CD146(+) SSCs were isolated using a magnetic activated cell sorting method. To quantitatively investigate the proliferation and differentiation ability, the colony-forming efficiency (CFE) and multi-lineage differentiation capacity of CD105(+) SSCs and CD146(+) SSCs were compared with unsorted cells and adipose-derived stem cells (ASCs). It was revealed that CD105(+) and CD146(+) subpopulations represented subsets of SSCs which generated chondrocytes and osteocytes, however not adipocytes. Compared with CD105(+) subpopulations and ASCs, the CD146(+) subpopulation exhibited a greater CFE and continuous high chondrogenic differentiation capacity in vitro. Therefore, the present study suggested that the CD146(+) subpopulation represented a chondrolineage-restricted subpopulation of SSCs and may therefore act as a valuable cell source for cartilage regeneration.