Abstract: This study verified the impact of Imb (intermuscular bone) deficiency on skeletal development in two ways: analyses of skeletal staining and the expression of skeletal development-related genes between wildtype and Imb mutant zebrafish. First, this study revealed the skeletal development of two Imb phenotypes from 8 dpf to 56 dpf. The results indicated that the development of most of the skeleton was synchronous except for the intermuscular bones. Furthermore, we detected the expression of 6 skeletal development-related genes (bmp2a, bmp4, smad1, smad4a, runx2a and sp7) in 5 embryonic stages (blastula, 3 hpf; gastrula, 6 hpf; segmentation, 12 hpf; pharyngula, 24 hpf; and the hatching period, 72 hpf) and 5 postembryonic stages (15 dpf, 30 dpf, 45 dpf, 60 dpf and 75 dpf). The results for the different embryonic stages showed that the expression of 4 genes (bmp2a, bmp4, smad1 and smad4a) in both wildtype and mutant individuals and the expression of sp7 in mutant individuals was increased in the blastula, gastrula and segmentation stages, then declined in the pharyngula and hatching period. However, the expression of runx2a in both wildtype and mutant individuals and the expression of sp7 in wildtype zebrafish continued to rise during embryonic development. In addition, there was no difference in the expression of the 6 genes between wildtype and mutant zebrafish in the blastula and gastrula stages, whereas a significant difference in the expression of the genes existed in the other 3 stages. The expression of bmp4, smad1, smad4a and runx2a in wildtype zebrafish was higher than that in mutant zebrafish, and the expression of sp7 in wildtype zebrafish was lower than that in mutant individuals, whereas there was no difference in bmp2a expression between wildtype and mutant individuals. Moreover, the results during postembryonic development indicated that the expression of the 6 genes declined significantly in 5 periods and that there was no difference between wildtype and mutant individuals. In summary, considering the results of the skeletal staining and gene expression analyses together, we speculate that the deletion of intermuscular bones does not impact the skeletal development of zebrafish.