Abstract: Inheritance of PGI isozymes in chestnut species was analyzed using isoelectric focusing on thin-layer polyacrylamide slab gels and single tree progeny method.Three alleles at one Pgi locus (Pgi-1) were found to be codominantly inherited.Two additional alleles were detected in samples of natural populations.Considerable variations in allele frequency and heterozygosity were found in populations of the American (Castanea dentata) and Chinese (C. mollissima) chestnut species.Heterozygosity at the Pgi locus was generally higher in Chinese than in American chestnut.Significantly higher heterozygosity of the Pgi was detected in the southernmost location of the natural range of the American chestnut.The highest and lowest heterozygosity was observed in Chinese chestnut populations from the Changjiang River and southeast region of China, respectively.