Background <p>Intermuscular bones (IBs) are segmental intramembranous ossifications located within myosepta. They share similarities with tendon ossification, a form of heterotopic ossification (HO). The mechanisms underlying IB formation remain incompletely understood.</p> Results <p>In this study, we systematically analyzed transcriptome data across multiple tissues, species, time points, and resolutions in teleosts. First, we identified IB-specific expression genes using the <i>tau</i> index method. Through cross-species comparisons of the tendon development process, we discovered that candidate genes were primarily enriched in extracellular matrix organization, ossification, regulation of angiogenesis, and other related processes. We also revealed that some of these candidate genes are abnormally expressed in <i>runx2b</i>^−/− zebrafish, which lack IBs. To clarify the trajectory of cell differentiation during IB formation, we demonstrated that myoseptal stem cells differentiate into osteoblasts, fibroblasts, and tenocytes in <i>runx2b</i>^+/+ zebrafish. However, in <i>runx2b</i>^−/− zebrafish, the differentiation of myoseptal stem cell into osteoblast was inhibited, while differentiation into <i>clec3bb</i> + tenocyte and fibroblast was enhanced. Additionally, <i>runx2b</i> deficiency led to the upregulation of <i>clec3bb</i> expression in the <i>clec3bb</i> + tenocyte cluster. Notably, a compensatory effect was observed in cell differentiation and gene expression in <i>runx2b</i>^−/− zebrafish, suggesting that <i>runx2b</i> and the candidate genes, such as <i>clec3bb</i>, were involved in the gene network of IB development.</p> Conclusions <p>The results elucidate cell differentiation process during tendon ossification in teleosts and identify the key factor <i>clec3bb</i> involved in this process. These findings provide a foundation for understanding tendon ossification in teleosts and for further research on tendon ossification in mammals.</p>