Otolith chemistry of young-of-year (YOY) yellowfin tuna Thunnus albacares was examined to determine whether chemical signatures are distinct across major spawning areas in the Atlantic Ocean. YOY yellowfin tuna otoliths were collected from 4 locations in the Atlantic Ocean (Gulf of Mexico, Caribbean Sea, Cape Verde, and Gulf of Guinea) from 2013-2015, and trace element (Li, Mg, Mn, Sr, Zn, and Ba) and stable isotope (delta C-13 and delta O-18) analyses were conducted to investigate regional variation in otolith chemical composition. Results indicated that significant regional differences in chemical signatures existed for each cohort of YOY yellowfin tuna investigated. Quadratic discriminant function analysis showed that nursery assignment accuracies based on otolith trace elements and stable isotopes were 64-85% for each cohort, justifying the use of these natural tracers as regional discriminators for yellowfin tuna. Significant interannual variability in regional signatures was also detected, highlighting the importance of age-class matching when using the baseline of nursery signatures to estimate the origin of sub-adult and adult yellowfin tuna. This study clearly demonstrates that baseline chemical signatures in the otoliths of YOY yellowfin tuna are distinct and can therefore serve as an effective tool for assigning older individuals to their nursery of origin, ultimately providing a way to improve our understanding of the population connectivity and mixing rates of this species in the Atlantic Ocean.