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To conserve and utilize the genetic pool of gynogenetic gibel carp (Carassius auratus gibelio), the Fangzheng and Qihe stock hatcheries have been established in China. However, little information is available on the amount of genetic variation within and between these populations. In this study, clonal diversity in 101 fish from these two stock hatcheries and 35 fish from two other hatcheries in Wuhan and Pengze respectively was analysed for variation in serum transferrin. Thirteen clones were found in Fangzheng and Qihe, of which 12 were novel. Six clones were specific to Fangzheng and three specific to Qihe, whereas four were shared among the Fangzheng and Qihe fish. To obtain more knowledge on genetic diversity and genealogical relationships within gibel carp, the complete mitochondrial DNA (mtDNA) control region (∼920 bp) was sequenced in 64 individuals representing all 14 clones identified in the four hatcheries. Differences in the mtDNA sequences varied remarkably among hatcheries, with the Fangzheng and Qihe lines demonstrating high diversity and Wuhan and Pengze showing no variation. The Fangzheng and Qihe lines might represent two distinct matrilineal sources. One of the Qihe samples carried the haplotype shared by a most widely cultivated Fangzheng clone, indicating that a Fangzheng clone escaped from cultivated ponds and moved into the Qihe hatchery. Four Fangzheng samples clustered within the lineage formed mainly by Qihe samples, most likely reflecting historical gene flow from Qihe to Fangzheng. It is suggested that clones in Wuhan originated from Fangzheng, consistent with their introduction history, supporting the hypothesis that gibel carp in Pengze were domesticated from individuals in the Fangzheng hatchery.