Abstract: The subfamily Amblyopinae of fish is one of the few marine bony fish that possess amphibious characteristics, primarily inhabiting intertidal mudflats. Compared with other amphibious fish with strong terrestrial capabilities, this group adopts a more passive terrestrial adaptation strategy, avoiding common terrestrial challenges by residing in mudflat burrows. However, the genetic mechanisms underlying its adaptation to mudflat burrowing remain unclear at present. This study conducted chromosome-level genome assembly and annotation of the Odontamblyopus lacepedii, a species of the subfamily Amblyopinae, using multi-platform sequencing data. Combined with comparative genomics analysis, it explored the phylogenetic relationships of the subfamily Amblyopinae and the molecular mechanisms underlying their adaptation to intertidal burrowing life. The results indicated that the genome size of O. lacepedii was 963.28 Mb, anchored to 23 chromosomes, with a total of 23558 protein-coding genes annotated. Phylogenetic analysis indicates that the subfamily Amblyopinae does not form an independent monophyletic group but is nested within the subfamily Oxudercinae, supporting a paraphyletic relationship and justifying the taxonomic viewpoint to merge the two groups. In comparison with four Oxudercinae species and seven additional outgroup identified 24 expanded and 243 contracted gene families, along with 163 positively selected genes in the Amblyopinae lineage. These genes were mainly enriched in signaling pathways related to olfactory perception, angiogenesis, and lipid/carbohydrate metabolism, which associated with their adaptation to the dark, hypoxic, and nutrient-poor cave environment in the mudflat. In addition, we identified 552 relaxation selection genes, a considerable portion of which are related to vision, pigmentation, and circadian rhythms, suggesting their role in the degeneration of traits such as visual degeneration, weakened body coloration, and loss of circadian rhythms in Amblyopinae. This study provides new insights into the environmental adaptation and key trait evolution mechanisms of the Amblyopinae and other intertidal burrowing fish.