Microsatellites are popular co-dominant markers for different genetic analyses, and transcriptome sequencing provides a new approach for rapid development of microsatellite markers. Gibel carp is a triploid gynogenetic fish with great growth potentials, but no transcript-associated microsatellite markers are available. Here we developed and characterized a set of EST-SSRs from the transcriptome data of gibel carp. 126 out of the 150 EST-SSRs were successfully developed and 110 showed polymorphisms in gibel carp. Twenty-four randomly selected polymorphic EST-SSRs were characterized in a cultured population of gibel carp, and the number of alleles per locus ranged from 3 to 9 (mean 6.5), with an average proportion of heterozygotes of 84.2%. All 24 EST-SSRs were successfully amplified in a goldfish family, and genotypes of all loci were in the Mendelian expectations after Bonferroni corrections. In addition, seven polymorphic EST-SSRs were selected for genetic diversity analysis in seven wild crucian carp populations of China, and the results showed that these loci have abundant polymorphisms with allele numbers of 13-37. Taken together with the genetic variations of crucian carp populations, the average number of alleles (N-a) was 20.9 per locus, and the mean H-o, H-e and PIC were 0.752, 0.878 and 0.865, respectively. Structure analysis and clustering results consistently supported that seven crucian carp populations could be divided into two major branches, and the genetic distances are basically positively correlated with geographical distances. These transcript-derived microsatellites are valuable resources and powerful tools for population genetics and many other genetic analyses in gibel carp and its close relatives.