Genomic DNA was extracted from heartwood blocks of six Cryptomeria japonica individuals that had been buried (in an area now covered by rice fields) for about 3600 years. Attempts were made to determine the sequences of five nuclear genes following polymerase chain reaction amplification, using previously obtained C. japonica expressed sequence tag (EST) information. We detected 15 nucleotide substitutions and four insertion/deletions (indels) in a partial GapC gene sequence among 13 individuals of the buried and an extant population, which allowed us to estimate the extent of DNA variation within the buried populations, and the level of genetic differentiation between the buried population and the extant population growing in a neighbouring area. For the entire haplotypes of the GapC region, π and θ nucleotide diversity estimates were 0.0063 and 0.0010, respectively, when both populations were included, while corresponding figures for the buried population alone were 0.0009 and 0.0017. Estimates of DNA divergence statistics (dXY = 0.0062, dA = 0.0005, FST = 0.0832 and KST = 0.0935) suggest that differentiation between the two populations was not great. However, permutation tests gave FST and KST values rejecting the null hypothesis (that populations were not differentiated) at the 5% and 1% probability levels, respectively. The significant genetic differentiation between the two populations was mainly caused by differences in haplotype diversity. The significant level of haplotype diversity in the extant population compared to the buried population might be the result of gene flow from neighbouring artificial forests. Alternatively, it is possible that we failed to detect all the DNA variation in the buried population because of clonal growth in the buried population.