Juglans regia L., Persian or English walnut, is an economically fruit crop species with edible kernels and widely planted in the temperate zones. Moldova has an ancient tradition in walnut cultivation increased during the centuries by a large spreading of seeds. Searching of walnut biotypes for establishing new varieties are important for future. Surveys of walnut germplasm were done in the northern (N), north west (NW) and central (C) agro ecological zones of the Republic of Moldova, and nuts were harvested on seventy trees of walnut from natural populations. Study focused on biodiversity evaluation through biochemical traits and genetic molecular markers has been the aim of the present research.  High differentiation between the three agro zones was observed for the biochemical traits while the molecular markers presented the Moldovan germplasm as a panmittic unit, indicating an environmental influences on kernel characteristics.