This article presents the first physical mapping carried out in the Senegalese sole (Solea senegalensis), an important marine fish species of Southern Europe. Eight probes were designated to pick up genes of interest in aquaculture (candidate genes) from a bacterial artificial chromosome (BAC) library using a method of rapid screening based on a 4-dimension PCR. Seven known and 3 unknown clones were isolated and labeled. The 10 BAC clones were used as probes to map the karyotype of the species by fluorescence in situ hybridization (FISH). Nine out of the 10 clones were localized in only 1 chromosome pair, whereas the remaining one hybridized on 2 chromosome pairs. The 2-color FISH experiments showed colocation of 4 probes in 2 chromosome pairs. In addition, 2-color FISH was carried out both with 5S rDNA and the BAC containing the lysozyme gene published previously. This first genetic map of the Senegalese sole represents a starting point for future studies of the sole genome. In addition, 7 out of the 10 BAC clones were sequenced using next-generation sequencing, and bioinformatic characterization of the sequences was carried out. Hence the anchoring of the sequences to specific chromosomes or chromosome arms is now possible, leading to an initial scaffold of the Senegalese sole genome.