Abstract: Intermuscular bone (IB) is a type of membrane bone that derive from the continuous homogeneous ossification of the connective tissue in fish muscle. Collagen genes involved in the regulation of vertebrate bone mineralization, but it is not clear whether its expression is related to the development of fish intermuscular bones. In order to study the correlation among collagen genes and IB formation, this study first analyzed the phylogenetic evolution of type Ⅰ and Ⅱ collagen genes in fish; then compared genes expression levels at different stages and tissues between Megalobrama amblycephala with intermuscular bones and Oreochromis niloticus without intermuscular bones. Phylogenetic analysis of the collagen genes col1a1, col1a2 and col2a1 showed that fish species with IB and species without IB were clustered into different branches, and the amino acid homology of M. amblycephala and O. niloticus genes was less than 90%. There were significant differences between the expression levels of col1a1a and col1a1b genes in M. amblycephala and homologous genes col1a1 in O. niloticus in several muscle (dorsal, upper caudal and lower caudal muscles). In M. amblycephala, the expression levels of type Ⅰ and type Ⅱ collagen genes in the dorsal muscle were higher than those in the caudal muscle; however, in O. niloticus, the expression of these genes was opposite to that of M. amblycephala. The expression of col1a1a and col2a1b genes of M. amblycephala increased significantly before the appearance of intermuscular bones (15 dph) and after the appearance of all intermuscular bones (50 dph). The relative expression of type Ⅰ collagen gene and col2a1b were significantly different in different periods, and the expression abundance of col1a1a at 50 dph was extremely high. Compared with M. amblycephala, the expression change of these genes in Oreochromis niloticus were small, and the overall fluctuation was not large. These results revealed that the expression level and pattern of col1a1 in two species were significantly different (P<0.01), therefore we speculate that col1a1 is potentially related to the development of IB.