Flow cytometry (FCM) has been used for plant DNA content estimation since the 1980s; however, presently, the number of laboratories equipped with flow cytometers has significantly increased and these are used extensively not only for research but also in plant breeding (especially polyploid and hybrid breeding) and seed production and technology to establish seed maturity, quality and advancement of germination. A broad spectrum of horticultural and medicinal species has been analyzed using this technique, and various FCM applications are presented in the present review. The most common application is genome size and ploidy estimation, but FCM is also very convenient for establishing cell cycle activity and endoreduplication intensity in different plant organs and tissues. It can be used to analyze plant material grown in a greenhouse/field as well as in vitro. Due to somaclonal variation, plant material grown in tissue culture is especially unstable in its DNA content and, therefore, FCM analysis is strongly recommended. Horticultural species are often used as internal standards in genome size estimation and as models for cytometrically studied cytotoxic/anticancer/allelopathic effects of different compounds. With the growing interest in genome modification, increased application of FCM is foreseen.