In this study, the presence and concentration of persistent organic pollutants (POP) were assessed in surface sediments collected from a wide variety of wetlands located throughout the Mekong Basin in Myanmar, Lao People’s Democratic Republic (PDR), Thailand, Cambodia, and Vietnam. Of the 39 POPs tested in 531 sediment samples, dichlorodiphenyltrichloroethane (DDT) and its metabolites endosulfan, hexachlorobenzene (HCB), and endrin were most commonly detected. Even though DDT was banned in the 1990s, some use of DDT may still be occurring in the Mekong Basin. The amount of metabolites for DDT—dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane (DDD)—found, however, suggests that use of DDT is on the decline throughout the region. HCB and endrin were found distributed broadly throughout the Mekong Basin but not in high amounts. The concentration and distribution of endosulfan and its metabolites represent a serious problem requiring further study and management action. While the total loading of POPs in wetland sediments of the Mekong Basin was generally low, hotspot sites occurred where concentrations exceeded established ecological risk thresholds. For example, wetlands of the open, dry dipterocarp forest of northern Cambodia and Vietnam as well as wetlands in the Mekong Delta of Vietnam contained high concentrations of some POPs. High concentrations of POPs were detected in some wetlands important for biodiversity conservation. Hotspots identified in wetlands such as the Tonle Sap not only had concentrations of DDT and DDE that exceeded Canadian and U.S. benchmarks, but fauna sampled in the area also showed high degrees of bioaccumulation of the same substances. Further and more extensive attention to monitoring POP presence in water birds, fish, and other aquatic organisms is warranted because of the bioaccumulation of these chemicals at higher levels in the food chain. This study represents a collaboration of eight universities from five countries in the Mekong Region (Myanmar, Lao PDR, Thailand, Cambodia and Vietnam) and four universities and research institutions from the United States. Funding for the study came from the Lower Mekong Initiative, U.S. Department of State.