Land use/cover change (LUCC) attributed to natural factors and human activities has led to the loss of ecosystem services, making the quantitative valuation of ecosystem services the scientific focal pointfor sustainable development. This study assessed changes in the ecosystem services values (ESVs) due to LUCC during the period 1990–2016 in the Kilombero Valley floodplain, located in southeastern Tanzania. Moderate resolution Landsat images from 1990, 2010 and 2016 were obtained and analyzed using a random forest (RF) algorithm for classification, and ArcGIS Desktop software (version 10.2, Esri, Redlands, CA, USA) for mapping to assess the LUCC. The ESVs were estimated based on the benefit transfer approach using adopted global value coefficients and modified local value coefficients. The results revealed that the aggregated ESVs of the forests, bushlands, wetlands, and water had decreased, consequently leading to a total loss of US$ 811.5 million (26.6%) in ESVs over the past 26 years when calculated with the modified local value coefficients to US$ 3000.7 million (42.3%) when calculated with global value coefficients. Moreover, the loss in the ESV was attributed to the decreased values of water regulation, climate regulation, erosion control, nutrient cyclying, habitat/refugia, and water supply, with the exception of the values of food production and biological control, which gradually increased during the study period. This study provided minimum estimates of the ecosystem service values, which willcontribute to the formulation of policy actions and strategies for sustainable management of the Kilombero Valley floodplain and inform various stakeholders on the tradeoffs involved in the use of land resources.