In this study, two mitotic gynogenesis families GF1 and GF2 were produced and verified with 10 microsatellite markers，8 in GF1 and 4 in GF2 respectively. The inheritance and segregation of 10 microsatellite loci in putative gynogenetic doubled haploids (GDH) were investigated. All of fries in control families were abnormal, while normal fries reappeared in GF1 and GF2 after hydrostatic pressure shock, with normality rate of 40.0% and 17.1% respectively. In GF1, twenty genotypes have been observed in 30 assayed progenies. All samples of GF1 were demonstrated as GDH for exclusive maternal inheritance and homozygous at each locus. In 30 tested offspring in GF2, 27 fries were demonstrated as GDH, 2 fries contained male parent specific band, and 1 fry remained undefined. These results suggested that the homozygous gynogenesis could be induced with the method reported in this paper. In addition, the segregations of microsatellite markers in GDHs were consistent with the expected ratio according to Mendel’s law at all the loci except LYC0026 and LYC0053. We also found that the segregation mode of GDH was completely identical between LYC0002 and LYC0014. In the present study, the artificial induction of homozygous gynogenesis and the inheritance and segregation of microsatellite markers in GDH in large yellow croaker was first reported, which will server as a foundation for rapid establishment of homozygous lines and making a genome map with GDH and homozygous lines in large yellow croaker.