The olive flounder (Paralichthys olivaceus) is an important cultured marine fish. However, little information is available on primordial germ cell (PGC) development and migration in this species; such information is vital for applications in artificial reproduction and for the preservation of genetic resources. Here, we sought to remedy this information deficit by isolating the germline-specific gene nanos3 and analyzing its expression in olive flounder. Sequencing analysis showed that olive flounder nanos3 contained a typical RNA-binding zinc finger domain. A phylogenetic analysis demonstrated that nanos3 of the olive flounder grouped with that of the barfin flounder (Verasper moseri). In the olive flounder, nanos3 was consistently expressed during embryogenesis. Whole-mount in situ hybridization showed that a new pattern of PGC migration was present in olive flounder, which combined elements of the PGC migration patterns of medaka, herring, and goby. In olive flounder, PGCs aligned along the lateral plate mesoderm in two loose, elongated lines at early embryogenesis, aggregated into a single loose cluster at mid-embryogenesis, then re-aligned into two tight clusters at late somitogenesis, and finally migrated to the genital ridge as two clusters. Furthermore, whole-mount in situ hybridization revealed that expression of stromal derived factor 1 (Sdf1) was important for guiding of PGC migration during somitogenesis. In particular, Sdf1 directed aggregation of PGCs into a single loose cluster from the two elongated lines during mid-embryogenesis. Additionally, PGCs in zebrafish were successfully visualized by injection of chimeric RNA containing the green fluorescent protein (GFP) and 3' untranslated region of olive flounder nanos3. These findings provide new insights into PGC migration and development in olive flounder and will also facilitate germ cell manipulation in this species.