Hybrid petunia cultivars are susceptible to infection by powdery mildew (Oidium longipes) and potyviruses (potato Y virus, petunia flower mottle virus) during commercial vegetative propagation. Plant proteins encoded by susceptibility genes are required by powdery mildew and potyviruses to infect plant hosts. To examine the potential of developing genetic resistance to these pathogens, petunia orthologs of the susceptibility genes MLO, eIF4E, and eIF(iso)4E were identified and characterized. High-resolution DNA melting analysis was conducted with a population of chemically-mutagenized petunia plants to identify mutations in these genes. Among 865 M2 families, point mutations were detected in coding regions of PhMLO1, but not eIF4E or eIF(iso)4E. One of the point mutations caused a glycine to glutamate substitution that was predicted to have a deleterious effect on PhMLO function. This study demonstrated the potential of TILLING for the development of pathogen resistance in petunia through the loss of susceptibility gene function.