The grass carp (Ctenopharyngodon idella) represents an economically important fish species in freshwater aquaculture within China. However, the urgency to develop superior varieties has become paramount due to significant germplasm degeneration in grass carp. Germline stem cell transplantation emerges as a promising and potent technique for reducing the sexual maturity cycle in fish. Consequently, our objective was to shorten the generation interval for obtain functional gametes of grass carp by employing barbel chub (Squaliobarbus curriculus) as a surrogate host. The accurate identification of germ cells between donor and recipient species is a critical step in the successful establishment of germline stem cell transplantation. However, the method of identification of germ cells in grass carp and barbel chub is not clear. In the present study, the full-length cDNA of reproduction-related gene nanos2 were cloned from grass carp and barbel chub, which are 649 and 636 base pairs, encoding 145 and 144 amino acids, respectively. Sequence analysis revealed that the grass carp Nanos 2 (CiNanos2) amino acid sequence exhibits a high degree of sequence identity to that of barbel chub (91.67%), and to that of zebrafish (Danio rerio) (65.49%). Phylogenetic tree analysis showed that grass carp is clustered together with barbel chub, indicating the closest genetic relationship between them. The expression of nanos2 transcripts in grass carp and barbel chub was predominantly observed in the gonads, with significantly higher levels detected in the testis compared to the ovary, suggesting that nanos2 might plays a crucial role in the development of gonad. Species-specific and common primers were designed based on the alignment of nanos2 sequences between grass carp and barbel chub for PCR analysis. The results demonstrated that the grass carp-specific primers had exclusively target product (179 bp) in the gonad of grass carp, while the barbel chub-specific primers had only expected product (265 bp) in the gonad of barbel chub gonads. Additionally, the common primers were able to amplify indiscriminately in the gonads of both grass carp and barbel chub, producing a target product of 251 bp, implying that the germ cells of grass carp and barbel chub could be efficiently distinguished via species-specific primers by PCR. Our study laid the foundation for further investigating the mechanism of the nanos2 gene in gonad development of grass carp and barbel chub. Meanwhile, it provided an effective method for monitoring the chimerism and development of grass carp germ cells in the gonads of barbel chub.