Nitrous oxide (NO) is an important greenhouse gas and an ozone-depleting substance which can be emitted from wastewater treatment systems (WWTS) causing significant environmental impacts. Understanding the NO production pathways and their contribution to total emissions is the key to effective mitigation. Isotope technology is a promising method that has been applied to WWTS for quantifying the NO production pathways. Within the scope of WWTS, this article reviews the current status of different isotope approaches, including both natural abundance and labelled isotope approaches, to NO production pathways quantification. It identifies the limitations and potential problems with these approaches, as well as improvement opportunities. We conclude that, while the capabilities of isotope technology have been largely recognized, the quantification of NO production pathways with isotope technology in WWTS require further improvement, particularly in relation to its accuracy and reliability.