The Senegalese sole (Solea senegalensis) is an economically important flatfish species. In this study, a genome draft was analyzed to identify microsatellite (SSR) markers for whole-genome genotyping. A subset of 224 contigs containing SSRs were preselected and validated by using a de novo female hybrid assembly. Overall, the SSR density in the genome was 886.7 markers per megabase of genomic sequences and the dinucleotide motif was the most abundant (52.4%). In silico comparison identified a set of 108 SSRs (with di-, tetra- or pentanucleotide motifs) widely distributed in the genome and suitable for primer design. A total of 106 markers were structured in thirteen multiplex PCR assays (with up to 10-plex) and the amplification conditions were optimized with a high-quality score. Main genetic diversity statistics and genotyping reliability were assessed. A subset of 40 high polymorphic markers were selected to optimize four supermultiplex PCRs (with up to 11-plex) for pedigree analysis. Theoretical exclusion probabilities and real parentage allocation tests using parent-offspring information confirmed their robustness and effectiveness for parental assignment. These new SSR markers were combined with previously published SSRs (in total 229 makers) to construct a new and improved integrated genetic map containing 21 linkage groups that matched with the expected number of chromosomes. Synteny analysis with respect to C. semilaevis provided new clues on chromosome evolution in flatfish and the formation of metacentric and submetacentric chromosomes in Senegalese sole.