During skeletal development, both osteogenic and chondrogenic programs are initiated from multipotent mesenchymal cells, requiring a number of signaling molecules, transcription factors, and downstream effectors to orchestrate the sophisticated process. Col10a1, an important downstream effector gene, has been identified as a marker for maturing chondrocytes in higher vertebrates, such as mammals and birds. In zebrafish, this gene has been shown to be expressed in both osteoblasts and chondrocytes, but no study has reported its role in osteoblast development. To initially delineate the osteogenic program from chondrogenic lineage development, we used the zebrafish col10a1 promoter to establish a transgenic zebrafish expressing a GFP reporter specifically in osteoblast-specific bone structures that do not involve cartilaginous programs. A construct harboring a -2.2-kb promoter region was found to be sufficient to drive the reporter gene in osteoblast-specific bone structures within the endogenous col10a1 expression domain, confirming that separable cis-acting elements exist for distinct cell type-specific expression of col10a1 during zebrafish skeletal development. The -2.2-kb col10a1:GFP transgenic zebrafish marking only bone structures derived from osteoblasts will undoubtedly be an invaluable tool for identifying and characterizing molecular events driving osteoblast development in zebrafish, which may further provide a differential mechanism where col10a1 is involved in the development of chondrocytes undergoing maturation in other vertebrate systems.