Tracer technologies based on naturally occurring substances or intentionally introduced compounds have a broad spectrum of applications in hydrogeological research and subsurface resource management. DNA (deoxyribonucleic acid)-based tracers, with unlimited unique variations and exceptional specificity, could potentially map the complex intricacies of subsurface flow networks in greater detail than traditional tracer methods. Here, we review recent advances in DNA-based tracer research involving modern culture-independent (i.e., molecular) measurement techniques for subsurface/flowpath characterization purposes. The two broad categories of DNA-based tracers, i.e., synthetic and naturally occurring, are further classified into four specific types: “naked DNA”, “encapsulated DNA”, “barcoding microbial communities”, and “indicator microbial communities”. We summarize and compare the basic methodological workflows for each type of DNA-based tracer and provide an overview of research developments in the past two decades, covering both laboratory/field-scale experiments and data interpretation methods. Finally, we highlight remaining questions and challenges for each type of DNA-based tracer in terms of practicality. Future research directions are also identified, including the application of emerging DNA tracer methods to a wider range of geological formations. Fundamental characteristics of these novel tracers need to be better understood, and their applicability under a broader range of engineering scenarios requires further validation.