Regardless of the high economic value and large geographical distribution of oysters, the current knowledge of oyster taxonomic relationships and systematics is still limited, particularly for flat oysters. In this study, the molecular cytogenetic characterization of mitotic chromosomes of the Provence flat oyster or dwarf oyster, Ostrea stentina, was performed through Giemsa staining, chromosome measurements, in situ restriction endonuclease banding, C-banding, fluorescence in situ hybridization with major ribosomal RNA genes (ITS1), and telomeric sequence (TTAGGG)_n. The karyotype (2n = 20) consisted of 6 metacentric (1, 3, 4, 6, 8, and 10) and 4 submetacentric (2, 5, 7, and 9) chromosome pairs. Chromosome treatment with HaeIII produced specific banding patterns for all chromosomal pairs, confirming the efficiency of this restriction enzyme for chromosome banding in oysters. Results for C-banding revealed the presence of heterochromatin in the telomeric regions of the short arms: on a large metacentric chromosomal pair and on a submetacentric chromosomal pair. In situ hybridization with telomeric sequence revealed bright hybridization signals in the telomeres of all chromosomes. The location of the major ribosomal rDNA (ITS1) displayed the presence of 2 signals: in the telomeric regions of the short arms of the largest metacentric chromosome and in a submetacentric chromosome. The cytogenetic data obtained was used to perform a comparative karyological analysis within the subfamily Ostreinae. It is also important to highlight that this type of work can provide new insights on major genomic changes at the chromosome level in the flat oysters.