Most of peach and nectarine cultivars are propagated traditionally on seedling rootstocks in Turkey. For this purpose, 'Uzunoglu' peach [Prunus persica L. (Batsch)] has been used extensively for long years. The fruits of 'Uzunoglu' peach are having no commercial value except good rootstock characteristics. The objectives of this study were to compare the isozyme polymorphism and nuclear DNA content variation in free-pollinated peach seedling populations. During the study, isozyme variation was surveyed for five enzymes including alcohol dehydrogenase (ADH, EC 1.1.1), isocitrate dehydrogenase (IDH, EC 220.127.116.11), malate dehydrogenase (MDH, EC 18.104.22.168), peroxidase (PRX, EC 22.214.171.124) and phosphogluco mutase (PGM, EC 126.96.36.199) in the seedlings. Moreover, genome size (C-value) was measured in peach seedling rootstocks. Cell nuclei were isolated from leaf tissues of the seedlings and then fluorescence intensities were measured on propidium iodide stained nuclei by using 'Tahiti' lime [Citrus latifolia Tan.] and mungbean [Vigna radiata 'Berken'] as internal reference standard by flow cytometry. According to the obtained results, isozyme variation was detected in 5 isozyme systems resulting 7 loci and 13 alleles. Isozyme polymorphism has only been observed in malate dehydrogenase profiles. Results obtained from flow cytometric analysis indicated that all analyzed seedlings were found to be diploid genomes with 0.574 pg/2C value in average. The study demonstrates the value of clonal rootstocks for eliminating of genetic variability within seedling rootstocks.