Cotton (Gossypium spp.) is the leading natural fiber crop in the world. The genus Gossypium comprises a broad genetic base that has been and continues to be actively studied by cotton genetists and breeders. Cytogenetic tools play an important role in cotton genome research and cotton breeding. Based essentially on the observation of chromosome morphologies and the analysis of chromosome pairing, classical cytogenetics has contributed greatly to understanding cotton history, taxonomy and phylogeny, and has been a great help in cotton breeding programs designed to transfer desired genes from alien species into cultivated varieties. With the advent of molecular cytogenetics in the 1980s, the field of cytogenetics has been revolutionized. Beside an increase in the speed, sensitivity and specificity of conventional cytogenetic techniques, molecular cytogenetics offers opportunities to perform a variety of tasks not achievable by classical methods. These tasks include analysis of the distribution of repeated sequences along the genome, assignment of repetitive and single copy DNA sequences to positions on chromosomes, determination of the relationship between specific chromosomes and linkage groups, determination of the relationships between physical and genetic distances, differentiation of the genomes involved in hybrids, detection of alien DNA in introgressed lines, and others. We summarize the achievements of classical and molecular cytogenetic investigations in Gossypium and underline the relevance of bridging these approaches in Gossypium genetic studies and exploitation.