PCR-based subtractive hybridization was used to isolate sequences from Erwinia amylovora strain Ea110, which is pathogenic on apples and pears, that were not present in three closely related strains with differing host specificities: E. amylovora MR1, which is pathogenic only on Rubus spp.; Erwinia pyrifoliae Ep1/96, the causal agent of shoot blight of Asian pears; and Erwinia sp. strain Ejp556, the causal agent of bacterial shoot blight of pear in Japan. In total, six subtractive libraries were constructed and analyzed. Recovered sequences included type III secretion components, hypothetical membrane proteins, and ATP-binding proteins. In addition, we identified an Ea110-specific sequence with homology to a type III secretion apparatus component of the insect endosymbiont Sodalis glossinidius, as well as an Ep1/96-specific sequence with homology to the Yersinia pestis effector protein tyrosine phosphatase YopH.