Aim: The present study was carried out to assess the effectiveness of pulp tissue of deciduous teeth stored in fresh water and natural soil, as a source of nuclear DNA and its use for the determination of gender using polymerase chain reaction (PCR). Materials and methods: One hundred and twenty extracted deciduous teeth were divided into two groups of 60 each and were stored in fresh water (group I) and natural soil (group II). These groups were divided into 3 subgroups (A, B, and C) of 20 each and stored for 3, 9, and 15 months, respectively, before subjecting for the PCR analysis. DNA was isolated and quantified. X and Y chromosomes were amplified by PCR and compared with the actual gender of the child. The data were analyzed using the Shapiro–Wilk test, the independent sample t-test, the paired t-test, and the chi-square test. Results: The pulp tissue of teeth stored in natural soil showed significantly higher correct interpretation than that of the teeth stored in fresh water. Conclusion: Even though the ability of determination of gender from the samples stored in fresh water and natural soil decreased with the prolongation of storage period, teeth stored in natural soil were found to be better than that of the ones stored in fresh water. Clinical significance: The pulp tissue of deciduous teeth is effective for gender determination and that would be extremely helpful for forensic experts for the identification of markedly skeletonized bodies.