Abstract: Supernumerary microchromosomes are non-essential chromosomes in addition to regular chromosomes, and it always believed that they have no biological functions. With the rapid development of sequencing technology, more and more evidence show that supernumerary microchromosomes have active genes and play important role in organism. Amphitriploid gibel carp (Carassius gibelio) and amphidiploid crucian carp (Carassius auratus) are belonged to the polyploid Carassius complex, and previous studies have shown that amphitriploid C. gibelio contain regular chromosomes and supernumerary microchromosomes while amphidiploid C. auratus are consist of regular chromosomes. To reveal genomic characteristics of the supernumerary microchromosomes in amphitriploid C. gibelio, we identified the potential sequences of supernumerary microchromosomes by supernumerary microchromosome-enriched satellite sequences and fluorescence in situ hybridization, and performed genomic characterization of these sequences. In this study, a total of 15 scaffolds were identified to be derived from supernumerary microchromosomes in the genome database of a female gibel carp, and 22.28% of these sequences had homologous to the regular chromosomes of amphidiploid C. auratus, which was the ancestral of gibel carp. The repetitive sequence proportion of supernumerary microchromosomes in amphitriploid C. gibelio was obviously higher than that of regular chromosomes in amphidiploid C. auratus and amphitriploid C. gibelio, and the contents of repetitive sequences were also different among them. Subsequently, we found that 6 of the 8 expanded meiosis-related gene families in C. gibelio had duplicated copies in the sequences of supernumerary microchromosomes, and these duplicated copies displayed transcriptional activity during oogenesis. In addition, we developed 3 SCAR markers specific to supernumerary microchromosomes based on these amplified copies, and these markers also could be used to distinguish amphidiploid C. auratus and amphitriploid C. gibelio. These results indicated that the supernumerary microchromosomes of amphitriploid C. gibelio originated from regular chromosomes, and accumulated numerous repeats and active genes during the evolution process. Besides, most gene expansions of meiosis-related genes were accomplished by supernumerary microchromosomes in C. gibelio. These findings not only revealed the genomic characteristics of supernumerary microchromosomes in amphitriploid C. gibelio, but also provided novel insight into the role of supernumerary chromosomes in the evolution of unisexual reproduction.