Abstract: Amphioxus has an important evolutionary position as a result of their phylogenetic position relative to vertebrates. Understanding their chromosomes would provide key points in the study of evolutionary biology and comparative genomics. The difficulty in preparing amphioxus chromosomes currently provides a significant hurdle in this research. In the current study, we describe an improved method for metaphase preparation from amphioxus embryos and methodology for preparing metaphase spreads from regenerative somatic cells. Chromosomes of two amphioxus species from Xiamen waters in China are also observed. The diploid chromosome number was found to be 40 in Branchiostoma belcheri, while B. japonicum has 36, confirming the two are distinct species from cytotaxonomic viewpoint.