A new technique was developed to study the cotton fiber initiation process and fiber initial densities. The goal was to provide an additional tool to cotton breeders and geneticists interested in fiber improvement. The objectives were to assess whether fiber initiation patterns reported for some Gossypium hirsutum L. and G. barbadense L. cultivars extend to a more diverse range of cultivars/lines; and to test if there is a relationship between fiber initials density, lint percentage, and fiber characteristics. This study was performed with 17 cultivars and experimental lines of G. hirsutum (upland cotton) and G. barbadense (Pima cotton) and one G. arboreum L. accession. The ovules were extracted from flowers at the day of anthesis and 1 and 2 d thereafter, stained with a fluorescent dye, DiOC6(3) (3,3′-dihexyloxacarbocyanine iodide), and observed under a light microscope. Staining cotton fiber initials with DiOC6(3) enabled the evaluation of a greater number of samples than the more time-consuming scanning electron microscopy protocols to assess the fiber initiation process. There were significant differences in fiber initial densities among cultivars and lines but these did not cluster into "upland" and "Pima" types as had been expected. Upland and Pima fiber initiation occurred at anthesis, whereas Sea Island Pima initiation was delayed a day. A delay in fiber initiation was also confirmed in fuzzless mutant lines. Fiber initial densities were not found to be useful predictors of lint percentage.