Abstract A project to research the application of nondestructive assay (NDA) to spent fuel assemblies is underway. The research team comprises the European Atomic Energy Community (EURATOM), embodied by the European Commission, DG Energy, Directorate EURATOM Safeguards; the Swedish Nuclear Fuel and Waste Management Company (SKB); two universities; and several United States national laboratories. The Next Generation of Safeguards Initiative–Spent Fuel project team is working to achieve the following technical goals more easily and efficiently than in the past using nondestructive assay measurements of spent fuel assemblies: (1) verify the initial enrichment, burnup, and cooling time of facility declaration; (2) detect the diversion or replacement of pins, (3) estimate the plutonium mass, (4) estimate the decay heat, and (5) determine the reactivity of spent fuel assemblies. This study focuses on spectrally resolved gamma-ray measurements performed on a diverse set of 50 assemblies [25 pressurized water reactor (PWR) assemblies and 25 boiling water reactor (BWR) assemblies]; these same 50 assemblies will be measured with neutron-based NDA instruments and a full-length calorimeter. Given that encapsulation/repository and dry storage safeguards are the primarily intended applications, the analysis focused on the dominant gamma-ray lines of 137 Cs, 154 Eu, and 134 Cs because these isotopes will be the primary gamma-ray emitters during the time frames of interest to these applications. This study addresses the impact on the measured passive gamma-ray signals due to the following factors: burnup, initial enrichment, cooling time, assembly type (eight different PWR and six different BWR fuel designs), presence of gadolinium rods, and anomalies in operating history. To compare the measured results with theory, a limited number of ORIGEN-ARP simulations were performed.