In this study, we analyzed fluctuating asymmetry (FA) of black locust (Robinia pseudoacacia) leaf traits as a measure of developmental instability in polluted and unpolluted habitats. We aimed to evaluate the potential of this method as a biomarker and its applicability on widely distributed species under in situ conditions. Leaf samples were taken from seven sites--three categorized as unpolluted (natural protected and rural) and four categorized as polluted covering the broad spectrum of intense pollution (industrial and traffic), from 1,489 individual trees in total. Results revealed significant differences in FA with expected higher values in polluted environments. Applicability of FA of R. pseudoaccacia leaf traits as a biomarker for testing potential pollution level, as well as the amount and distribution of sampling effort needed for its application, are discussed.