High-Mobility Group (HMG) B proteins are abundant and highly conserved non-histone proteins, which play an important architectural role in the assembly of nucleoprotein complexes and in the Regulation of transcription. These proteins have also been shown to play key roles during embryonic development and cell differentiation. Here we report a full-length cDNA sequence of the HMG protein, AmphiHMGB from Amphioxus. Sequence analysis indicates that this putative AmphiHMGB protein contains four domains: HMG-box A, HMG-box B, basic region, acidic carboxyl-terminal tail and a linker. Phylogenetic analysis suggests that AmphiHMGB falls outside the vertebrate clade. HMGB gene duplication occurred near the base of the vertebrate gene Clade. The dynamic expression of AmphiHMGB during embryonic development reveals for the first time that it may involve differentiation of neural ectoderm, mesoderm and endoderm in this animal.