We evaluated type of stomata, as well as differences in stomatal frequency in three areas of the plant (apical, middle and basal) under two culture conditions (in vitro and ex vitro), in four cactus species (Opuntia ficus-indica, Hylocereus undatus, Escontria chiotilla and Mammillaria petterssonii). We measured stomatal density (SD) and the stomata index (SI) for both factors, plant area and culture condition. Stomata typology were cyclocytic, tetracytic or opuntioid type in O. ficus-indica and parallelocytic type in E. chiotilla, H. undatus and M. petterssonii. It was observed that SD was higher ex vitro than in vitro in H. undatus and M. petterssonii, but the opposite was true for O. ficus-indica and E. chiotilla. The apical region had the highest SD both in in vitro and ex vitro, and the interaction between culture condition and explant areas was highly significant, implying that the impact of culture type is different for each distinct stem area.