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Synopsis How animal embryos determine their early cell fates is an important question in developmental biology. In various model animals, asymmetrically localized maternal transcripts play important roles in axial patterning and cell fate specification. Cephalochordates (amphioxus), which have three living genera (Asymmetron, Epigonichthys, and Branchiostoma), are an early branching chordate lineage and thus occupy a key phylogenetic position for understanding the evolution of chordate developmental mechanisms. It has been shown that in the zygote of Branchiostoma amphioxus, which possesses bilateral gonads flanking both sides of their trunk region, maternal transcripts of germline determinants form a compact granule. During early embryogenesis, this granule is inherited by a single blastomere, which subsequently gives rise to a cluster of cells displaying typical characteristics of primordial germ cells (PGC). These PGCs then come to lie in the tailbud region and proliferate during posterior elongation of the larvae to join in the gonad anlagen at the ventral tip of the developing myomeres in amphioxus larvae. However, in Asymmetron and Epigonichthys amphioxus, whose gonads are present only on the right side of their bodies, nothing is known about their PGC development or the cellular/morphogenetic processes resulting in the asymmetric distribution of gonads. Using conserved germline determinants as markers, we show that similarly to Branchiostoma amphioxus, Asymmetron also employs a preformation mechanism to specify their PGCs, suggesting that this mechanism represents an ancient trait dating back to the common ancestor of Cephalochordates. Surprisingly, we found that Asymmetron PGCs are initially deposited on both sides of the body during early larval development; however, the left-side PGCs cease to exist in young juveniles, suggesting that PGCs are eliminated from the left body side during larval development or following metamorphosis. This is reminiscent of the PGC development in the sea urchin embryo, and we discuss the implications of this observation for the evolution of developmental mechanisms.