Objective To identify the drop-off location of victims in drowning cases, and confirm whether it is a fatal drowning or the victim is thrown into the water after death by detecting part of 5.8S sequence and second internal transcribed spacer （ITS2） （5.8S+ITS2） of diatom rDNA in water and organs. Methods Two cases identified by diatom examination, which received by Nanjing Municipal Public Security Bureau Forensic Center, were taken as the research objects. The difference of the population structure of algae in water and human tissue was analysed by length polymorphism of 5.8S+ITS2 marker. Results In case 1, similar species of diatom were detected from victim’s lung and liver tissues and the water sample. Two kinds of DNA fragments with length of 330 bp and 376 bp were detected from victim’s lung tissue and the water sample using 5.8S+ITS2 marker, which could confirm the victim was drowning before death. In case 2, there was no diatom found in victim’s lung and liver tissues. Only one kind of DNA fragment with length of 331 bp and low relative fluorescence unit （RFU） was obtained from victim’s lung tissue using 5.8S+ITS2 marker, thus the victim was thrown into the water after death. Conclusion The experimental results of the two cases in present study are consistent with the actual facts and the result of the diatom microscopic examination. The difference of population structure of specific microorganism in water and human tissue can be detected by 5.8S+ITS2 marker, which can help to identify the drop-off location of victims in drowning cases, and confirm whether it is a fatal drowning or the victim is thrown into the water after death.