BAC-FISH (bacterial artificial chromosome and fluorescence in situ hybridization) is a powerful tool for molecular cytogenetics such as chromosome identification and physical mapping in plant. Due to the large proportion of repetitive sequence occurring in some plant species，especially the polyploidy species, the application of BAC-FISH was hindered severely. Molecular markers from a high-density genetic map of tetraploid cotton were used to screen BAC libraries. Protocols of BAC-FISH in cotton mitotic and meiotic cells were introduced by some modifications such as root tip preparation, slide denaturation and post-hybridization washing according to previous protocols. The dual-color, dual-probe and repeated FISH were also introduced by applying them to physical mapping a random BAC clone on the corresponding chromosome.