The top priority of zoos and aquariums is the welfare of the animals in their immediate care. Environmental factors have the potential to be stressful and negatively affect the welfare of animals housed in human care; therefore, it is crucial to monitor the well-being of animals who are asked to cope with environmental changes. The aim of this study was to investigate the welfare of the ambassador African penguin colony at the Columbus Zoo & Aquarium before and after the introduction of a new group of penguins and to a new habitat by assessing personality, novel object test responses, behavior, and urofecal glucocorticoid metabolite (ufGCM) levels. The study subjects consisted of 9 penguins, 4 females and 5 males, ranging from 3 to 27 years old. All data were collected in two phases (P1 and P2), representing the periods before and after the environmental changes were implemented. P1 and P2 personality results were similar, both accounting for 95% of the variance in the data set and consisting of an Unsociable, Curious, and Aggressive components. When comparing the two phases, the majority of novel object test responses were highly correlated. Both of these results suggest the penguins personalities were unaffected by the environmental changes. The behavioral and ufGCM data showed a quick acclimation to the environmental changes; however, there was considerable individual variation in this response. This research helps the Columbus Zoo & Aquarium uphold their standard of making management decisions backed with scientific evidence to give their animals the best opportunity for optimal welfare. Future studies should attempt to measure the effect of an individual management style based on personality assessments versus a group management style on the impact of environmental stressors.