Molecular cytogenetic techniques, especially Fluorescence In situ Hybridization (FISH) and Genomic In situ Hybridization (GISH), are excellent tools to study the structure and function of chromosomes, polyploidy, aneuploidy, alien gene introgression and genome evolution and physical mapping of genes. For many applications, in situ hybridizations require reliable and efficient methods of chromosome preparation with well preserved and dispersed chromosomes and little or no cell wall debris. However, such protocols have not yet been published for cotton mitotic chromosome preparations. In the current study, an improved method for preparing mitotic metaphase chromosomes of tetraploid cotton was developed. Root tips were collected from lab-grown three-day-old cotton seedlings and pre-treated with high-pressure nitrous oxide (N2O). Following fixation with acetic acid, root tip meristems were removed, enzymatically digested, gently macerated, and then processed to create suspensions of protoplasts in acetic acid and methanol. Suspended protoplasts were then dropped onto a glass slide in order to distribute the protoplasts and spread the chromosomes. Using this approach, acceptable mitotic indexes and high-quality chromosomal spreads with no cell wall debris were obtained. This drop method of preparing chromosome spreads was tested for its compatibility for in situ hybridization using rDNA as probe in FISH. Additionally, varying pre-treatment times with N2O were investigated. Our results show that the combination of a 95-minute N2O pre-treatment, a 55-minute enzymatic degradation, and the "drop method" efficiently produce cotton mitotic metaphase preparations. This new method will expectedly improve the efficiency and efficacy of GISH, FISH, and single-copy multicolor FISH, which will have a multitude of ramifications in cotton genetics and breeding.