Grass carp (Ctenopharyngodon idellus, GC), as the highest-output fish in China, is economically important. The production of gynogenetic grass carp (GGC) will provide important germplasm resource for producing improved GC. At present, knowledge regarding the heterologous sperm DNA in gynogenetic offspring is little. Thus, revealing paternal DNA in GGC at the molecular level would be highly significant for fish genetic breeding. In this study, ultraviolet-treated sperm of koi carp (Cyprinus carpio haematopterus, KOC, 2n = 100), was used to activate the eggs of GC (2n = 48). Afterwards, cold shock (0–4 °C) was administered for 12 min to double the chromosomes, resulting in GGC. No significant difference (p > 0.05) was found between GGC and GC in appearance, erythrocytes size and chromosome numbers. However, at the molecular level, a specific microsatellite DNA fragment (MFW1-gynogenetic grass carp, MFW1-G) derived from the paternal parent KOC was found to be transmitted into GGC. For the first time, this study provided an evidence at the molecular level that the DNA fragment derived from the paternal parent occurred in GGC. This finding is of great significance for fish genetic breeding.