Among chordate taxa, the cephalochordates diverged earlier than urochordates and vertebrates; thus, they retain unique, primitive developmental features. In particular, the amphioxus notochord has muscle-like properties, a feature not seen in urochordates or vertebrates. Amphioxus contains two genes, and . is reportedly expressed in the blastopore, notochord, somites, and tail bud, in contrast to a low level of expression only in notochord. To distinguish the expression profiles of the two genes at the single-cell level, we carried out single-cell RNA-seq (scRNA-seq) analysis using the amphioxus, . This scRNA-seq analysis classified embryonic cells into 15 clusters at developmental stages from midgastrula to early swimming larva. was expressed in cells of clusters 4, 5, 8, and 9. We first confirmed that cluster 8 comprises cells that form somites since this cluster specifically expresses four myogenic factor genes. Cluster 9 contains a larger number of cells with high levels of expression and a smaller number of cells with expression. Simultaneous expression in cluster 9 of tool-kit genes, including , , and , showed that this cluster comprises cells that form the notochord. Expression of , but not , in cells of clusters 4 and 5 at the gastrula stage together with expression of and indicates that clusters 4 and 5 comprise cells of the blastopore, which contiguously form the tail bud. In addition, , and were highly expressed in -expressing clusters 4, 5, 8, and 9 in a temporally coordinated manner, suggesting roles of anterior Hox genes in specification of mesodermal organs, including somites, notochord, and tail bud. This scRNA-seq analysis therefore highlights differences between the two genes in relation to embryonic regions in which they are expressed and their levels of expression. is the ancestral in amphioxus, since expression in the blastopore is shared with other deuterostomes. On the other hand, is a duplicate copy and likely evolved a supplementary function in notochord and somite formation in the lineage.