Cotton (Gossypium, Malvaceae) has been spun, woven and dyed since prehistoric times. Four cotton species are economically important; these are Gossypium arboreum (tree cotton), G. barbadense (American pima cotton), G. herbaceum (levant cotton) and G. hirsutum (American upland cotton). Previous research has been conducted examining the pollen grains of the Malvaceae and there is a key that differentiates the four economically important species of Gossypium by their pollen grains. However, the cotton pollen found in boll weevils, Anthonomus grandis Boheman, and other insect pests cannot be keyed to the species using the published key. The objective of this research was to determine if the pollen grains of these four species could be differentiated and develop a key that works for cotton pollen found in insect pests. Flowers of the four taxa were collected from USDA greenhouses and fields and dried. Both unacetolysed and acetolysed pollen grains were examined with light and scanning electron microscopy. The length and width of 300 pollen grains and 100 processes (spines) of each taxon were measured. There were no size differences between the acetolysed and the unacetolysed grains. Gossypium barbadense and G. hirsutum represent the largest grains (mean =106.7 and 94.9 µm, respectively) and the longest processes (mean= 17.7 and 15.2 µm, respectively). Differentiation of these taxa can be useful in the determination of the origin of insect pests that attack cotton when different cotton species are grown within a region. However, additional research is needed on the varieties and cultivars of cotton.