Ex-situ preservation of genetic resources is an essential strategy for the conservation of plant biodiversity. In this regard, seed storage is the most convenient and efficient way of preserving germplasm for future plant breeding efforts. A better understanding of the molecular changes that occur during seed desiccation and aging is necessary to improve conservation protocols and real-time methods for monitoring seed quality. Epigenetic regulation plays an important role in the management of plant growth, development, and response to stress factors. Several reports have indicated that DNA methylation plays a critical role in seed development and viability. This study examines changes in 5-methylcytosine (5mC) levels in the DNA of seeds during aging, a process that has important implications for plant conservation and agriculture. The changes in the level of genomic 5-methylcytosine (5mC) in seeds of Populus nigra L. by 2D-TLC were assessed. P. nigra is a riparian forest species. The riparian forest species are a key priority in biodiversity conservation and climate change strategies. Therefore, there is an interest in protecting and preserving P. nigra germplasm as it is a pioneer species and a key component of softwood forests in Europe. In our study, we demonstrate for the first time that 5mC levels decrease during storage and that the decline can be detected before any changes in seed germination are evident. Once P. nigra seeds reached an 8-10% reduction in the level of 5mC, a substantial decrease in germination occurred. The decline in the level of 5mC appears to be a critical parameter underlying the rapid deterioration of intermediate seeds. Thus, the measurement of 5mC can be a fast, real-time method for assessing asymptomatic aging in stored seeds.