Few studies have reported the use of toothbrushes as a reliable source of DNA for human or gender identification. The present systematic review with the available information was conducted to answer the focus question “Is a toothbrush a reliable source of DNA for human or gender identification?”. The keyword combination “Toothbrush” and “DNA” was used to search databases including MEDLINE, Scopus, and Web of Science along with a manual search of reference lists of relevant articles. Duplicates and irrelevant articles were excluded, and the remaining articles were fully read for the final selection of articles. The risk of bias of the included studies was evaluated using the Appraisal tool for Cross-Sectional Studies (AXIS tool). Of the 130 articles obtained, 122 duplicates or irrelevant articles were eliminated. Following the full-text reading of eight articles, five articles were selected based on eligibility criteria. The five studies reported that a toothbrush is a good source of DNA irrespective of the time interval. In a few studies some samples were not sufficient for complete DNA profiling due to factors such as the method of DNA extraction. Although a toothbrush is an excellent source of DNA for human and gender identification, future studies with a larger sample size, appropriate control group, and standardized technique of DNA extraction need to be conducted. Additionally, factors influencing the quantity and quality of DNA in toothbrushes need to be determined with standardized techniques.