Tropospheric ozone is one of the most reactive air pollutants, which causes visible injuries, as well as biomass and yield losses. The negative effect of ozone is cumulative during the growing season; hence crops are the most sensitive plants. Visible symptoms and biomass losses can cause economic losses. Tobacco plants have been recognized as one of the best bioindicators, but data on the cumulative effect of ozone on this species are limited. Results of an experiment with ozone-sensitive tobacco plants grown on sites varying in ozone concentration are presented in this paper. Two indices were used for data presentation of visible leaf injury degree. Higher solar radiation was the main cause of higher ozone concentration at the rural site. Higher tropospheric ozone concentrations were noted in 2010 in comparison to 2011, which was reflected in visible leaf injury. Canonical variate analysis did not reveal highly significant differences between sites, however, differences were observed in certain investigation periods. Moreover, higher leaf injury was noted at the rural site at the end of the experiment in both experimental years. This indicates the cumulative effect of ozone during the growing season. However, higher injury variability was noted at the urban site, even though lower ozone concentrations were noted there. Lower variability of injury at the rural site might suggest lack of influence of particulate matter and occurrence of higher injury even though lower ozone concentrations occurred. Better detection of ozone injury was shown by the first index based on three mean values.