Little information is available on the process of DNA release from plants and neither is there much information to be found regarding DNA transport in the vadose zone. Unsaturated soil columns were used to examine the release and transport of DNA content in the leaf of tomato variety Palmiro, which was introduced into the soil columns after being dried at 35 °C for 3 days. Soil columns were leached with sterile water at a rate of 0.5 mL h−1 for 104 days. DNA from column leachate water was extracted and analysed. Both quantitative and qualitative measurements of extracted DNA were taken. Tomato gene sequences were detected using PCR amplification based on tomato specific 18S primers. The concentration of total DNA in soil column leachate water ranged from 6.7 to 50.4 μg L−1. Genomic analysis using agarose gel electrophoresis showed degradation of DNA after its passage through unsaturated soil columns. The presence of tomato genes in the leachate water of soil columns suggests a potential release of tomato DNA from leaves and potential transport of DNA over considerable distances in water-unsaturated soil. Consequently, transport of plant DNA in vadose zone is indicated, and there is a risk that the DNA may reach the groundwater.