Local germplasm may be the basis for genetic improvement for sustainability and resilience, yet little is known about Greek peach [Prunus persica (L.) Batsch] local genetic resources. The aims of the present study were to entail a prospection in the mainland and islands of Greece for local traditional and underutilized germplasm and study their genetic and morphological variation and susceptibility to shoot blight from Cylindrocarpon destructans and Monilinia laxa. A total of 32 peach cultivars/accessions were prospected from the mainland (Imathia and Magnesia) and islands (Andros, Ikaria, Kythira, Lesvos and Samos) of Greece. Leaf and fruit morphological characterization was made in situ and ex situ using 42 qualitative and quantitative morphological descriptors. Nine clones of ‘Lemonato’, isolated in Magnesia, with differing ripening times, were found to vary in leaf and fruit phenotypic traits. The local peach genotypes were separated from 12 old foreign cultivars, in 8 out of the 42 leaf and fruit phenotypic traits studied. We observed greater lesion damages from C. destructans than M. laxa. Local cultivars had greater resistance to Cylindrocarpon destructans than foreign cultivars, with ‘Lemonato Andrea’, ‘Daggalakou’ and ‘Papagianni’ exhibiting the greatest resistance. Genetic characterization was performed in the studied local and foreign peach cultivars/accessions, using eight Inter Simple Sequence Repeats (ISSRs), resulting in a total of 404 bands. Analysis of molecular variance and principal coordinates analysis revealed moderate to low genetic diversity among the peach cultivars, and three distinct clusters were formed. Furthermore, multiple regression analysis was implemented for the association study between morphological traits and the ISSR markers, revealing several markers that are statistically and significantly correlated with fruits’ traits. The obtained results could be valuable for breeding programs and future research on peach.