Zhikong scallop (Chlamys farreri) is a bivalve species with broad economic and biological value, and an essential species of aquaculture in North China. Recently, efforts have been made to improve knowledge of genome, genetics, and cytogenetics, which is devoted to develop the molecular breeding project for the scallop. In this study, we constructed a cytogenetic map and identified all chromosomes of C. farreri using fluorescence in situ hybridization (FISH). A total of 100 Bacterial Artificial Chromosome (BAC) clones and 27 fosmid clones, including 58 microsatellite marker-anchored BAC clones, 4 genes-anchored BAC clones, 38 random BAC clones, 22 repetitive sequences-anchored fosmid clones, and 5 gene-anchored fosmid clones, were tested as probes, and 69 of them produced specific and stable signal on one pair of chromosomes. Then, multiple co-hybridizations were conducted to distinguish all the submetacentric and subtelocentric chromosomes with similar morphology by the abovementioned chromosome-specific markers. On this basis, a cytogenetic map of C. farreri containing 69 clones was constructed by co-hybridization and karyotype analysis. The markers covered all 19 pairs of chromosomes, and the average number of markers on each chromosome was 3.6. The cytogenetic map provides a platform for genetic and genomic analysis of C. farreri, which facilitates the molecular breeding project of C. farreri and promotes the comparative studies of chromosome evolution in scallops and even bivalves.