Sweet chestnut (CastaneasativaMill.) is a species that has been cultivated and propagated through grafting for a long time in Italy and Southern European countries. The genetic variability within and among different varieties was analyzed by means of starch gel electrophoresis. Twenty cultivated varieties originating from three different areas located in northern, central, and southern Italy, were analyzed at six polymorphic enzyme gene loci. Our results show a relatively high degree of homogeneity both among individuals of the same variety and among varieties of the same area; on the other hand, high values of genetic distance were found among different geographic areas. The agamic propagation method of this species may have caused a reduction of the genetic diversity within varieties. The causes and consequences of the loss of genetic variation in these varieties are discussed.