Cortisol is recognized as a physiological indicator of stress in fish. However, this hormone is typically measured in plasma samples. In this study, cortisol content was evaluated for the first time in the scales of tropical tuna. The sample included 20 skipjack tuna (Katsuwonus pelamis) and 25 yellowfin tuna (Thunnus albacares) collected in the Atlantic (the Gulf of Guinea off the Ivory Coast) and the Indian Ocean (off Reunion Island), and their scales were analyzed using an ELISA test. The results indicated that on average, cortisol concentration (1) did not show any geographical pattern, (2) was independent of fish size, and (3) was significantly higher in skipjack (mean 4.75 ± 6.56 ng g−1) than in yellowfin (mean 1.65 ± 1.85 ng g−1), although the difference was mainly due to four skipjack individuals. Larger datasets would be needed to confirm any species difference in cortisol concentration. The particularly high cortisol concentration observed in four individuals may be due to fish from different schools, suggesting the need for future sampling from free-swimming schools to investigate intra- and inter-school variability in cortisol concentration. This study opens the door for future research, including the collection of scales on tagged individuals, to investigate the links between chronic stress and behavior in these species.