One goal for evolutionary biology is to decipher the mystery underlying morphological novelties and complexity of extant organisms, especially vertebrates. As the largest and most diverse lineage in chordate, Vertebrata is characterized by several key morphological characteristics such as a tripartite brain. Such morphological characteristics are usually originated from variation of the related genetic toolkits. Hence, expression pattern and evolutionary analyses of key genes in these toolkits can deepen our understanding of the emergence of morphological innovations during evolution. In this study, we first analyzed the molecular evolution of khdrbs gene family, and then investigated zebrafish khdrbs genes expression patterns during embryonic development and in adulthood of zebrafish. The results indicated that after vertebrate WGD, khdrbs gene family was co-opted for brain development, and the duplication and diversification of khdrbs genes may promote the origin of vertebrate elaborate brains during evolution.