Trees were surveyed in 1994 and 1995 in southern Ontario for symptoms of leaf scorching and infection by Xylella fastidiosa. Samples were collected from 114 trees in 48 locations, and DNA was extracted from leaf veins and petioles of affected leaves. The DNA was used in the polymerase chain reaction (PCR) with primers that amplify a 733 base-pair fragment from isolates of X. fastidiosa. Several samples yielded PCR products of varying sizes, but only three samples from American elm (Ulmus americana) yielded the 733 base pair fragment characteristic of X. fastidiosa that also hybridized with the PCR product of a strain isolated from a tree infected with the bacterium. These trees were all located within the Niagara Peninsula. This is the first report of X. fastidiosa in Ontario and represents the most northerly distribution of X. fastidiosa-infected trees in eastern North America.