The distribution of corn stunt spiroplasma (Spiroplasma kunkelii, class Mollicutes, family Spiroplasmataceae, CSS) in its leafhopper vector Dalbulus maidis (DeLong & Wolcott) was studied by immunofluorescence confocal laser scanning microscopy (iCLSM), by using three preparation methods: dissected whole-mount leafhopper organs, thick sections of paraffin-embedded leafhopper parts, and smears of leafhopper hemocytes. These methods combined revealed the localization of CSS in the midgut, filter chamber, Malpighian tubules, hindgut, fat tissues, hemocytes, muscles, tracheae, and in several lobes of the salivary glands. However, CSS was not detected in nerve cells of the brain or other nerve ganglia of CSS-infected D. maidis. The percentage of leafhoppers with detected CSS in various organs/tissues 2 to 3 wk post-exposure to CSS-infected plants for 1 wk was up to 95%, and 75% of these leafhoppers inoculated CSS into maize, Zea mays L., test seedlings before iCLSM processing. Using thick sections from CSS-infected maize leaves, iCLSM was also used to localize CSS in phloem tissues of maize plants. Although transmission electron microscopy (TEM) provides higher resolution for the localization of mollicutes or other pathogens in host plants and insect vectors/hosts, iCLSM has the following advantages compared with TEM: 1) processing and examination of specimens is considerably faster; 2) much larger and more numerous samples can be processed; and 3) the distribution of pathogens can be studied in whole organs or even the entire insect (through thick sectioning). Additionally, compared with epifluorescence microscopy, iCLSM provides three-dimensional images of the studied organs, indicating their spatial relationships, which can be valuable for studying the routes of pathogens in their vectors.