Twist genes are essential for embryonic development and are conserved from jellyfish to human. To study the vertebrate twist family and its evolution, the entire complement of twist genes was obtained for 9 representative species. Phylogenetic analysis showed that a single protochordate twist gene was duplicated at least twice before the teleost-tetrapod split to give rise to 3 ancestral genes, which were further duplicated or deleted, resulting in fluctuating number of twist paralogs in different vertebrate lineages. To find whether changes in gene copy number were associated with changes in gene function, embryonic expression patterns of twist orthologs were evaluated against the number of twist paralogs in different species. The results showed evidence for both neo- and subfunctionalization, and, in addition, for loss of an ancestral regulatory gene. For example, in Xenopus, twist2 was lost, but the twist1 paralog acquired, and therefore preserved, twist2 function. A general model is proposed to explain the data. In this process, termed synfunctionalization, one paralog acquires the expression domain(s) of another. The merging may lead to function shuffle. Alternatively, it may leave one paralog redundant and thus subject to deletion--while its function is retained by the surviving paralog(s). Synfunctionalization is a mechanism that, together with neo- and subfunctionalization, may work to establish equilibrium in the number of genes that regulate developmental processes; it may regulate the complexity of regulatory regions as well as gene copy number and therefore may play a role in evolution of gene function and the structure of genome.