Description: Background: High-resolution cytogenetic map can provide not only important biological information on genome organization but also solid foundation for genetic and genomic research. The progress in the molecular and cytogenetic studies has created the basis for developing the cytogenetic map in cucumber (Cucumis sativus L.). Results: Here, the cytogenetic maps of four cucumber chromosomes (chromosomes 1, 3--5) were constructed by fluorescence in situ hybridization (FISH) analysis on cucumber pachytene chromosomes. Together with our previously constructed cytogenetic maps of three cucumber chromosomes (chromosomes 2, 6--7), cucumber has a complete cytogenetic map with 76 anchoring points between the genetic, the cytogenetic and the draft genome assembly maps. To compare our pachytene FISH map directly to the genetic linkage and draft genome assembly maps, we used a standardized map unit---relative map position (RMP) to produce the comparative map alignments. The alignments allowed a global view of the relationship of genetic and physical distances along each cucumber chromosome, and accuracy and coverage of the draft genome assembly map. Conclusions: We demonstrated a good correlation between positions of the markers in the linkage and physical maps, and essentially complete coverage of chromosome arms by the draft genome assembly. Our study not only provides essential information for the improvement of sequence assembly but also offers molecular tools for cucumber genomics research, comparative genomics and evolutionary study.