Abstract: This study investigated genetic diversity and genetic structure of six wild crucian carp populations collected from Heilongjiang River, Yangtze River, Fenghua River and Huaihe River by 8 genome microsatellites (G-SSR) and 8 transcriptome microsatellites (EST-SSR). The mean N_a, N_e, H_o, H_e and PIC of the eight G-SSR loci were 22, 12.9, 0.769, 0.893 and 0.879, respectively. F_st values ranged from 0.008 to 0.085, indicating that there were different degrees of genetic differentiation among the six wild crucian carp populations, among which two populations from the Heilongjiang River system were different from all the others. There was no significant genetic differentiation among the four populations in the Yangtze River, Fenghua River and Huaihe River. The Nei’s genetic distances of six wild crucian carp populations were between 0.203—0.701. UPGMA cluster analysis and Bayesian analysis supported that six crucian carp populations could be divided into two large branches with two populations from Heilongjiang water system as one group while the others as another group. The average N_a, N_e, H_o, H_e and PIC of the eight EST-SSR loci were 19, 9.5, 0.728, 0.870 and 0.855 respectively. The F_st values and Nei’s genetic distances among the six populations were between 0.005—0.084 and 0.117—0.683, respectively. Genetic differentiation analysis, UPGMA cluster analysis and Bayesian analysis based on EST-SSR markers also separated six populations of crucian carp into two major groups: the Heilongjiang water system group as well as the Yangtze River, Fenghua River and the Huaihe River system group. Based on G-SSR and EST-SSR markers, the average polymorphism information content (PIC) of the six crucian carp populations ranged between 0.786—0.864 and 0.761—0.833, respectively, indicating that all the six wild crucian carp populations had a high genetic diversity. Although the polymorphism of the EST-SSR markers was slightly lower than that of the G-SSR markers, the two types of SSR markers revealed similar genetic structure and pattern of differentiation for crucian carp in three major waters of China. This study provides basic and new information for the conservation of crucian carp germplasm resources and the evaluation of EST-SSR markers in genetic studies of fish populations.