There are two kinds of male in the breeding populations of the half-smooth tongue sole Cynoglossus semilaevis, namely natural males (ZZ) and neomales (ZW). It is of great importance to study their phenotypic and genetic characteristics since the knowledge can improve its breeding techniques and seed cultivation. In this study, 15 phenotypic traits were measured between mature ZZ males and ZW males (sex-inverted males) of C. semilaevis. Analysis showed that highly significant differences between ZZ males and ZW males existed in seven traits, including body weight, full length, body length, body width, head length, eye spacing and eye diameter, with the growth rate of ZW males being higher than that of ZZ males. Furthermore, comparative analysis of the population genetic structures was processed among maternal half-sib families between ZZ and ZW males by using 50 pairs of specific microsatellite markers. Results showed that 22 out of 50 pairs of PCR amplified markers were polymorphic. Among four half-sib families, 232 alleles were identified with relatively higher mean allelic number (Na) and mean expected heterozygosity (He) compared to mean effective allelic number (Ne) and observed heterozygosity (Ho) respectively. In addition, the average polymorphism information content (PIC) was 0.4566, 0.4254, 0.5069, 0.4795 among four half-sib families and there were 3, 3, 4 and 5 loci significantly deviated from Hardy-Weinberg expected genotypic frequencies. The Nei´s genetic distance among the four families was 0.2101–0.5840, and the genetic similarity was 0.5577–0.8105. The current study indicated that there were significant differences among growth traits between ZZ and ZW males of C. semilaevis. Between the maternal half-sib families, there was relatively high genetic similarity, low genetic distance and similar genetic diversity index.