Background: Members of the eukaryotic translation initiation complex are co-opted in viral infection, leading to susceptibility in many crop species, including stone fruit trees (Prunus spp). Therefore, modification of one of those eukaryotic translation initiation factors or changes in their gene expression may result in resistance. Objective: We searched the crop and wild Prunus germplasm from the Armeniaca and Amygdalus taxonomic sections for variants in the eIF4E and eIFiso4E genes, to identify alleles potentially linked to resistance to the Plum Pox Virus (PPV). Methodology/Results: Over one thousand stone fruit accessions (1,397) were screened for variation in eIF4E and eIFiso4E transcript sequences which are in single copy within the diploid Prunus genome. We identified new alleles for both genes that are not evident in haplotypes associated with PPV susceptible individuals. Overall, analyses showed that eIFiso4E is genetically more constrained since it displayed less polymorphism than eIF4E. We also demonstrated more variation at both loci in the related wild species than in crop species. As the eIFiso4E translation initiation factor was identified as indispensable for PPV infection in Prunus species, a selection of ten different eIFiso4E haplotypes along 13 individuals were tested by infection with PPV and eight of them displayed a range of reduced susceptibility to resistance, indicating new potential sources of resistance to sharka.