Stomatal density (SD) and stomatal conductance (gs) can be affected by an increase of atmospheric CO2 concentration. This study was conducted on 17 species growing in a naturally enriched CO2 spring and belonging to three plant communities. Stomatal conductance, stomatal density and stomatal index (SI) of plants from the spring, which were assumed to have been exposed for generations to elevated CO2, and of plants of the same species collected in a nearby control site, were compared. Stomatal conductance was significantly lower in most of the species collected in the CO2 spring and this indicated that CO2 effects on gs are not of a transitory nature but persist in the long term and through plant generations. Such a decrease was, however, not associated with changes in the anatomy of leaves: SD was unaffected in the majority of species (the decrease was only significant in three out of the 17 species examined), and also SI values did not vary between the two sites with the exception of two species that showed increased SI in plants grown in the CO2‐enriched area. These results did not support the hypothesis that long‐term exposure to elevated CO2 may cause adaptive modification in stomatal number and in their distribution.