Tunicates, the sister clade of vertebrates, have miniature genomes and numerous intronless genes compared to other animals. It is still unclear how the tunicates acquired such a large number of intronless genes. Here, we analyzed sequences and intron–exon organizations of homologous genes from two closely related tunicates, Ciona intestinalis and Ciona savignyi. We found seven cases in which ancestral introns of a gene were completely lost in a species after their divergence. In four cases, both the intronless copy and the intron-containing copy were present in the genome, indicating that the intronless copy was generated by retroduplication. In the other three cases, the intron-containing copy was absent, implying it was lost after retroduplication. This result suggests that retroduplication and loss of parental genes is a major mechanism for the accumulation of intronless genes in tunicates.