We sequenced at mRNA level in adult hearts of zebrafish, pamprey and sea squirt. Combined with other 11 vertebrate heart RNA-Seq data online, we conducted comprehensive evolutionary genomic analyses to address the contribution of gene/genome duplications on heart structure evolution. We observed that number of duplicate genes expressed in heart increased gradually with the increase of heart chamber number along the vertebrate phylogeny, despite that most of them were duplicated at the time near to the origin of vertebrates or more ancient. Our research provides a clear-cut example to show the relationship among gene duplication, continuous character evolution like heart structure evolution and nature selection. Overall design: Examination of mRNA levels in thirteen vertebrate samples with different cardiac chamber number, and one invertebrate (sea squirt).