As the third biggest genus in Sapindaceae, Acer contains about 110 species which are mainly distributed in the temperate regions of the Northern Hemisphere. With nine native species in Iran, maples are considered to be among the key forest trees in the country. The aim of this study is to assess the potential of petiole traits for delimiting species of Acer. Ten petiolar characters were studied in 44 populations of 9 species of Acer from Iran. The characters were assembled into a matrix and the ability of variables to segregate taxa was analyzed statistically. Results revealed a high reliability in delimiting all the species investigated. Acer monspessulanum, which occupies various habitats throughout the country, showed the highest diversity in petiole anatomy among the species studied. Based on the presence or absence of medullary bundles, two supertypes of petioles were definable. Although not helpful at the infraspecific level, petiole anatomy is shown to be taxonomically useful in delimiting species of Acer.