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Simple Summary: Rice-fish farming refers to the cultivation of fish in rice fields. China's Guizhou Province has a long history (over 1000 years) of Rice-fish farming. Carp (golden-backed carp) that have been cultivated for generations in rice fields have evolved to be extremely adaptable to that environment. Unfortunately, farmers and enterprises have found that the feeding ability and growth rate of golden-backed carp are becoming increasingly weaker and slower, respectively. Therefore, the present study carried out to examine the population genetics of golden-backed, and the results indicated that the status of germplasm resources of this species is not very good. The aim of this study was to further elucidate the status of golden-backed carp germplasm resources in this province. In conclusion, this work can provide reliable fundamental data for the future conservation and breeding of golden-backed carp. The aim of this study was to assess the current status of the germplasm resources of golden-backed carp (Cyprinus carpio var. Jinbei) cultured in paddy fields in Guizhou Province, China. Five populations of golden-backed carp in Liping County, Jinping County, Huangping County, Congjiang County and Duyun City in Guizhou Province were subjected to high-throughput sequencing by 2b-RAD technology, and their genetic diversity and genetic differentiation were analysed. Based on sequencing, 44,896 SNP loci were obtained, and all five population genetic diversity indicators showed low diversity. In the NJ tree, the Congjiang and Liping populations were mixed together, and the other three groups formed a cluster. A cross-validation error box plot and pong cluster plot were constructed to show the K value results. When K = 1, the cross-validation error rate was the lowest. Principal component analysis showed that the Duyun population formed a group separate from the group comprising the other four populations. The genetic differentiation index and genetic distances between the Duyun population and the remaining four populations were greater than 0.05, indicating population differentiation. The genetic diversity of the five populations of golden-backed carp in Guizhou Province was low, the genetic differentiation of the Duyun population was the most significant, and the Duyun population was separate from the other four groups. [ABSTRACT FROM AUTHOR]